Immunogenic epitopes for immunotherapy

ABSTRACT

The present invention relates to peptides, nucleic acids and cells for use in immunotherapeutic methods. In particular, the present invention relates to the immunotherapy of cancer. The present invention furthermore relates to tumor-associated T-helper cell peptide epitopes, alone or in combination with other tumor-associated peptides that serve as active pharmaceutical ingredients of vaccine compositions which stimulate anti-tumor immune responses. The present invention relates to novel peptide sequences and their variants derived from HLA class I and class II molecules of human tumor cells which can be used in vaccine compositions for eliciting anti-tumor immune responses.

RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No.12/180,045, filed on Jul. 25, 2008 and claims priority to Europeanapplication number EP07014797.0, filed on Jul. 27, 2007 and U.S.Provisional application 60/953,161, filed on Jul. 31, 2007 which areboth incorporated by reference in their entirety. For the purposes ofthe present invention, all references as cited herein are incorporatedby reference in their entireties.

FIELD OF THE INVENTION

The present invention relates to novel amino acid sequences of peptidesderived from tumour associated antigens that are able to bind to MHCcomplexes of either class, and elicit an immune response.

BACKGROUND OF THE INVENTION

Stimulation of an immune response is dependent upon the presence ofantigens recognised as foreign by the host immune system. The discoveryof the existence of tumour associated antigens has now raised thepossibility of using a host's immune system to intervene in tumourgrowth. Various mechanisms of harnessing both the humoral and cellulararms of the immune system are currently being explored for cancerimmunotherapy.

Certain elements of the cellular immune response are capable ofspecifically recognising and destroying tumour cells. The isolation ofcytotoxic T-cells (CTL) from tumour-infiltrating cell populations orfrom peripheral blood suggests that these cells play an important rolein natural immune defences against cancer (Cheever et al., Annals N.Y.Acad. Sci. 1993 690:101-112; Zeh H J, et al.; J. Immunol. 1999,162(2):989-94). CD8-positive T-cells (TCD8⁺) in particular, whichrecognise Class I molecules of the major histocompatibility complex(MHC)-bearing peptides of usually 8 to 10 amino acid residues derivedfrom proteins or defect ribosomal products (DRIPS) (Schubert U, et al.,Nature 2000; 404(6779):770-774) located in the cytosol, play animportant role in this response. The MHC-molecules of a human are alsodesignated as human leukocyte-antigens (HLA).

There are two classes of MHC-molecules: MHC class I molecules that canbe found on most cells having a nucleus that present peptides thatresult from proteolytic cleavage of endogenous proteins, DRIPS, andlarger peptides. MHC class II molecules can be found predominantly onprofessional antigen presenting cells (APCs), and present peptides ofexogenous proteins that are taken up by APCs during the course ofendocytosis, and are subsequently processed (Cresswell P., Annu Rev.Immunol. 1994; 12:259-93). Complexes of peptide and MHC class Imolecules are recognised by CD8-positive cytotoxic T-lymphocytes bearingthe appropriate TCR, and complexes of peptide and MHC class II moleculesare recognised by CD4-positive-helper-T-cells bearing the appropriateTCR. It is well known that the TCR, the peptide and the MHC are therebyabundant in a stoichiometric amount of 1:1:1.

CD4-positive helper T-cells play an important role in orchestrating theeffector functions of anti-tumour T-cell responses. For this reason, theidentification of CD4-positive T-cell epitopes derived from tumourassociated antigens (TAA) may be of great importance for the developmentof pharmaceutical products for triggering anti-tumour immune responses(Kobayashi, H., et al., 2002. Clin. Cancer Res. 8:3219-3225; Gnjatic,S., et al., 2003. Proc. Natl. Acad. Sci. U.S.A. 100(15):8862-7). CD4+ Tcells can lead to locally increased levels of IFNγ, a criticalrequirement of interferon gamma-mediated angiostasis for tumourrejection by CD8+ T cells (Qin Z, et al., Cancer Res. 2003 J;63(14):4095-4100).

In the absence of inflammation, expression of MHC class II molecules ismainly restricted to cells of the immune system, especially professionalantigen-presenting cells (APC), e.g., monocytes, monocyte-derived cells,macrophages, dendritic cells. In tumour patients, cells of the tumourhave surprisingly been found to express MHC class II molecules (DengjelJ, et al., Clin Cancer Res. 2006; 12:4163-4170).

It was shown in mammalian animal models, e.g., mice, that even in theabsence of CTL effector cells (i.e., CD8-positive T lymphocytes),CD4-positive T-cells are sufficient for inhibiting visualization oftumours via inhibition of angiogenesis by secretion of interferon-gamma(IFNγ) (Qin, Z. et al., 2000. Immunity. 12:677-686). Additionally, itwas shown that CD4-positive T-cells recognizing peptides fromtumour-associated antigens presented by HLA class II molecules cancounteract tumour progression via the induction of antibody (Ab)responses (Kennedy, R. C., et al., Cancer Res. 63:1040-1045). Incontrast to tumour-associated peptides binding to HLA class I molecules,only a small number of class II ligands of TAA have been described sofar. See generally, the syfpeithi database listing known MHC ligands andpeptide motifs and Cancer Immunity, the Journal of Academy of CancerImmunology.

Since the constitutive expression of HLA class II molecules is usuallylimited to cells of the immune system (Mach, B., et al., 1996. Annu Rev.Immunol. 14:301-331), the possibility of isolating class II peptidesdirectly from primary tumours was not considered possible. However,Dengjel et al. were recently successful in identifying a number of MHCClass II epitopes directly from tumours (See EP 04 023 546.7, EP 05 019254.1; Dengjel J, et al., Clin Cancer Res. 2006; 12:4163-4170).

For a peptide to trigger (elicit) a cellular immune response, it mustbind to an MHC-molecule. This process is dependent on the allele of theMHC-molecule and specific polymorphisms of the amino acid sequence ofthe peptide. MHC-class-I-binding peptides are usually 8-10 amino acidresidues in length and usually contain two conserved residues(“anchors”) in their sequence that interact with the correspondingbinding groove of the MHC-molecule. In this way each MHC allele has a“binding motif” determining which peptides can bind specifically to thebinding groove (Rammensee H. G., et al, Chapman & 1998 Hall MHC Ligandsand Peptide Motifs).

In the MHC class I dependent immune reaction, peptides not only have tobe able to bind to certain MHC class 1 molecules expressed by tumourcells, they also have to be recognized by T-cells bearing specificT-cell receptors (TCR).

The antigens that are recognised by the tumour specific cytotoxicT-lymphocytes, that is, their epitopes, can be molecules derived fromall protein classes, such as enzymes, receptors, transcription factors,etc., which are up-regulated in cells of the respective tumour.Furthermore, tumour associated antigens, for example, can also be uniqueto tumour cells, for example as products of mutated genes or fromalternative open reading frames (ORFs), or from protein splicing(Vigneron N, et al., Science 2004 Apr. 23; 304 (5670):587-90). Anotherimportant class of tumour associated antigens are tissue-specificantigens, such as CT (“cancer testis”)-antigens that are expressed indifferent kinds of tumours and in healthy tissue of the testis.

Various tumour associated antigens have been identified. Further, muchresearch effort has been spent to identify additional tumour associatedantigens. Some groups of tumour associated antigens, also referred to inthe art as tumour specific antigens, are tissue specific. Examplesinclude, but are not limited to, tyrosinase for melanoma, PSA and PSMAfor prostate cancer and chromosomal cross-overs such as bcr/abl inlymphomas. However, many tumour associated antigens identified to dateoccur in multiple tumour types, and some, such as oncogenic proteinsand/or tumour suppressor genes (tumour suppressor genes are, for examplereviewed for renal cancer in Linehan W M, et al., J. Urol. 2003December; 170(6 Pt 1): 2163-72), which actually cause the transformationevent, occur in nearly all tumour types. For example, normal cellularproteins that control cell growth and differentiation, such as p53(which is an example for a tumour suppressor gene), ras, c-met, myc,pRB, VHL, and HER-2/neu, can accumulate mutations resulting inup-regulation of expression of these gene products thereby making themoncogenic (McCartey et al. Cancer Research 1998 15:58 2601-5; Disis etal. Ciba Found. Symp. 1994 187:198-211). These mutant proteins can alsobe a target of a tumour specific immune response in multiple types ofcancer.

For proteins to be recognised by cytotoxic T-lymphocytes astumour-specific or -associated antigens, and for them to be used in atherapy, particular prerequisites must be fulfilled. The antigen shouldbe expressed mainly by tumour cells and not, or in comparably smallamounts, by normal healthy tissues. It is furthermore desirable, thatthe respective antigen is not only present in a type of tumour, but alsoin high concentrations (i.e. copy numbers of the respective peptide percell). Tumour-specific and tumour-associated antigens are often derivedfrom proteins directly involved in transformation of a normal cell to atumour cell due to a function e.g. in cell cycle control or apoptosis.Additionally, downstream targets of the proteins directly causative fora transformation may be upregulated and thus may be indirectlytumour-associated. Such indirectly tumour-associated antigens may alsobe targets of a vaccination approach (Singh-Jasuja H., et al., CancerImmunol. Immunoether. 2004 March; 453 (3): 187-95). In both cases it isessential to have epitopes in the amino acid sequence of the antigen,since such peptide (“immunogenic peptide”) that is derived from a tumourassociated antigen should lead to an in vitro or in vivoT-cell-response.

Basically, any peptide able to bind a MHC molecule may function as aT-cell epitope. A prerequisite for the induction of an in vitro or invivo T-cell-response is the presence of a T-cell with a correspondingTCR and the absence of immunological tolerance for this particularepitope.

Therefore, TAAs are a starting point for the development of a tumourvaccine. The methods for identifying and characterizing the TAAs arebased on the use of CTL that can be isolated from patients or healthysubjects, or they are based on the generation of differentialtranscription profiles or differential peptide expression patternsbetween tumours and normal tissues (Lemmel C., et al., Nat. Biotechnol.2004 April; 22(4):450-4; T. Weinschenk, et al., Cancer Res. 62(20):5818-5827, 2002.).

However, the identification of genes overexpressed in tumour tissues orhuman tumour cell lines, or selectively expressed in such tissues orcell lines, does not provide precise information as to the use of theantigens transcribed from these genes in an immune therapy. This isbecause only an individual subpopulation of epitopes of these antigensare suitable for such an application since a T-cell with a correspondingTCR has to be present and immunological tolerance for this particularepitope needs to be absent or minimal. It is therefore important toselect only those peptides from overexpressed or selectively expressedproteins that are presented in connection with MHC molecules againstwhich a functional T-cell can be found. Such a functional T-cell isdefined as a T-cell that upon stimulation with a specific antigen can beclonally expanded and is able to execute effector functions (“effectorT-cell”).

T-helper cells play an important role in orchestrating the effectorfunction of CTLs in anti-tumour immunity. T-helper cell epitopes thattrigger a T-helper cell response of the T_(H1) type support effectorfunctions of CD8-positive killer T-cells, which include cytotoxicfunctions directed against tumour cells displaying tumour-associatedpeptide/MHC complexes on their cell surfaces. In this waytumour-associated T-helper cell epitopes, alone or in combination withother tumour-associated peptides, can serve as active pharmaceuticalingredients of vaccine compositions which stimulate anti-tumour immuneresponses.

Since both types of response, CD8 and CD4 dependent, contribute jointlyand synergistically to the anti-tumour effect, the identification andcharacterization of tumour-associated antigens recognised by either CD8+CTLs (ligand: MHC class I molecule+peptide epitope) or by CD4-positiveCTLs (ligand: MHC class II molecule+peptide epitope) is important in thedevelopment of tumour vaccines. It is therefore an object of the presentinvention, to provide novel amino acid sequences for peptides that areable to bind to MHC complexes of either class.

SUMMARY OF THE INVENTION

The present invention relates to peptides, nucleic acids and cells foruse in immunotherapeutic methods. In particular, the present inventionrelates to the immunotherapy of cancer. The present inventionfurthermore relates to tumour-associated T-helper cell peptide epitopes,alone or in combination with other tumour-associated peptides that serveas active pharmaceutical ingredients of vaccine compositions whichstimulate anti-tumour immune responses. The present invention relates tonovel peptide sequences and their variants derived from HLA class I andclass II molecules of human tumour cells which can be used in vaccinecompositions for eliciting anti-tumour immune responses.

One embodiment of the invention provides peptides comprising a sequencethat is selected from the group of SEQ ID NO: 1 to SEQ ID NO: 29 or avariant thereof which is 80% homologous to SEQ ID NO: 1 to SEQ ID NO: 29or a variant, which will induce T cells cross-reacting with saidpeptide. In some embodiments, the peptides or variants have an overalllength of between 8 and 100, preferably between 8 and 30, and mostpreferably between 8 and 16 amino acids. The peptides preferably havethe ability to bind to a molecule of the human major histocompatibilitycomplex (MHC) class-I or -II. The present invention also providespeptides that consist of or consist essentially of an amino acidsequence according to SEQ ID NO: 1 to SEQ ID NO: 29. The peptides may bemodified, including modifications that include non-peptide bonds. Thepresent invention also provides fusion proteins comprising the peptides,in particular comprising N-terminal amino acids of the HLA-DRantigen-associated invariant chain (Ii).

The present invention also provides nucleic acids encoding the peptidesof the invention. The nucleic acid may be DNA, cDNA, PNA, CNA, RNA orcombinations thereof. Another embodiment of the invention comprisesexpression vectors capable of expressing the nucleic acids of theinvention. Host cells comprising nucleic acids or expression vectors ofthe present invention are also contemplated. The host cell may be anantigen presenting cell, in particular a dendritic cell.

Another embodiment also provides a peptide, nucleic acid, or expressionvector of the present invention for use in medicine.

The present invention also provides methods for producing a peptide ofthe invention by culturing a host cell of the invention and isolatingthe peptide from the host cell or its culture medium.

The present invention also provides in vitro methods for producingactivated cytotoxic T lymphocytes (CTL). The methods comprise contactingin vitro CTL with antigen loaded human class I or II MHC moleculesexpressed on the surface of a suitable antigen-presenting cell for aperiod of time sufficient to activate the CTL in an antigen specificmanner. The antigen is a peptide of the present invention. The antigenmay be loaded onto class I or II MHC molecules expressed on the surfaceof a suitable antigen-presenting cell by contacting a sufficient amountof the antigen with an antigen-presenting cell. The antigen-presentingcell may comprise an expression vector capable of expressing a peptidecontaining SEQ ID NO 1 to SEQ ID NO 29 or a variant thereof.

The present invention also provides activated cytotoxic T lymphocytes(CTL) produced by the methods described above, which selectivelyrecognise a cell that aberrantly expresses a polypeptide comprising anamino acid sequence of the present invention.

Another embodiment of the present invention also provides methods ofkilling target cells in a patient in which target cells aberrantlyexpress a polypeptide comprising an amino acid sequence of the presentinvention. The method comprises administering to the patient aneffective number of cytotoxic T lymphocytes (CTL) as described above.

The present invention also provides use of any peptides, nucleic acids,expression vectors, or activated cytotoxic T lymphocytes according tothe invention as a medicament or in the manufacture of a medicament. Themedicament may be a vaccine and may be active against cancer. The cancermay be glioblastoma, colorectal, pancreatic, lung, renal or gastriccancer.

The present invention further provides a kit comprising peptides of thepresent invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the ESI-liquid chromatography mass spectra identifyingtumour associated peptide (TUMAP) PCN-002 from colon carcinoma sampleCCA707 (FIG. 1 a), TOP-002 from glioblastoma sample GB 1006 (FIG. 1 b),PTP-001 from glioblastoma sample GB 1006 (FIG. 1 c), GAL-001 from renalcell carcinoma sample RCC 190 (FIG. 1 d), CHI-001 from glioblastomasample GB 1002 (FIG. 1 e), JAK-001 from glioblastoma sample GB 1002(FIG. 1 f), AKR-001 from non-small cell lung cancer NSCLC-Pool 2 (FIG. 1g), and FNI-001 from pancreatic carcinoma sample PC330 (FIG. 1 h) thatwere presented in a MHC class I restricted manner.

FIG. 2 shows the ESI-liquid chromatography mass spectra identifyingtumour associated peptide (TUMAP) CEA-009 from gastric carcinoma GC-Pool2 (FIG. 2 a), TGFBI-006 from gastric carcinoma GC-Pool 1 (FIG. 2 b),TGFBI-007 from glioblastoma sample GB6002 (FIG. 2 c), TGFBI-008 fromglioblastoma sample GB1004 (FIG. 2 d), TGFBI-009 from non-small celllung cancer NSCLC-Pool 1 (FIG. 2 e), and TGFBI-010 from glioblastomasample GB6002 (FIG. 2 f) that were presented in a MHC class IIrestricted manner.

FIG. 3 depicts the expression profiles of two genes encodingglioblastoma associated peptides PTP-001 (FIG. 3 a) and CHI-001 (FIG. 3b). Expression of the genes is absent or very low in normal tissueswhile increased up to more than 250-fold in glioblastoma samples(GB1006T to GB1011T; NCH359T and NCH361T).

FIG. 4 depicts binding affinities of selected peptides to HLA-A*0201 asmeasured by EpI ELISA according to Sylvester-Hvid, C, et al., 2002,Tissue Antigens, 59, 251-258). The analysis was limited to peptidesknown to be MHC class I binding peptides. Affinities of HLA-DR bindingpeptides cannot be measured with this assay.

FIG. 5 depicts the Tetramer analysis of microsphere driven proliferationof ODC-001 and NOX-001 specific CD8+ lymphocytes from peripheral blood.1×10⁶ CD8+ enriched PBMCs per well of the healthy HLA-A*0201+ donorHD100 was stimulated weekly with microspheres coupled to anti-CD28 plushigh density tumor antigen A*0201/ODC-001 (upper panel) or anti-CD28plus high density tumor antigen A*0201/NOX-001 (lower panel). Afterthree stimulations in vitro, all cells were stained with antibody CD8FITC plus tetramers A*0201/NOX-001 PE and A*0201/ODC-001 APC. Cells aregated on the lymphocyte population or CD8+ lymphocytes (right panel) andnumbers represent percentage of tetramer+ within CD8+ lymphocytes.

FIG. 6 depicts the in vitro immunogenicity of TGFBI-004 as detected byIFNγ ELISPOT after five stimulation cycles.

Cells were primed and restimulated repeatedly with TGFBI-004 and thenincubated with relevant TGFBI-004 (Well 1, 2, 3 and 4) and irrelevant(Neg. control) peptide, respectively. The analysis after IFNγ ELISPOTwas performed on an ELISPOT Reader (CTL, Cleveland, USA). PHA-Ionomycinserved as positive control. Numbers indicate the count of positivespots.

FIGS. 7 a and 7 b depict the in vitro immunogenicity of TGFBI-004 asdetected by ICS after five stimulation cycles.

Cells were primed with TGFBI-004-loaded autologous DCs and restimulatedrepeatedly with autologous PBMCs plus TGFBI-004. For the read-out cellswere incubated with relevant TGFBI-004 (Well 1, 2, 3 and 4) andirrelevant (Neg. Control) peptide, respectively. Additionally to theintracellular IFNγ staining, cells were also stained with CD4-FITC andOCD8-PerCP antibodies. The analysis was performed on a four-colorFACSCalibur cytometer (BD Biosciences, Germany).

FIG. 8 depicts the ELISPOT analysis of IFNγ production by T-cell linesupon in vitro restimulation with the NOX-001 peptide. A. T-Cell line 7+from donor HBC-154 (sorted CD8+ NOX-001 tetramer+); B. T-Cell line 7−from donor HBC-154 (sorted CD8+ NOX-001 tetramer-).

Sorted CD8+ NOX-001 tetramer+ (A.) and CD8+ NOX-001 tetramer− (B.) cellswere analysed by IFNγ ELISPOT after restimulation with irrelevant(MLA-001) (upper wells) and relevant (NOX-001) (lower wells) peptide (10μg/ml). Numbers indicate the count of positive spots.

FIG. 9 shows the results of tests where affinities of peptides containedin the present invention to HLA-A*0201.

DETAILED DESCRIPTION OF THE INVENTION

In a first aspect the invention provides a peptide comprising a sequencethat is selected from the group of SEQ ID NO: 1 to SEQ ID NO: 29 or avariant thereof which is 80% homologous to SEQ ID NO: 1 to SEQ ID NO: 29or a variant, which will induce T-cells cross-reacting with saidpeptide.

In the present invention, the term “homologous” refers to the degree ofidentity between sequences of two amino acid sequences, i.e. peptide orpolypeptide sequences. The aforementioned “homology” is determined bycomparing two sequences aligned under optimal conditions over thesequences to be compared. The sequences to be compared herein may havean addition or deletion (for example, gap and the like) in the optimumalignment of the two sequences. Such a sequence homology can becalculated by creating an alignment using, for example, the ClustalWalgorithm (Nucleic Acid Res., 22(22): 4673 4680 (1994). Commonlyavailable sequence analysis software, more specifically, Vector NTI,GENETYX or analysis tools provided by public databases, may also beused.

A person skilled in the art will be able to assess whether T-cellsinduced by a variant of a specific peptide will be able to cross-reactwith the peptide itself by (Fong, L, et al., Proc. Natl. Acad. Sci.U.S.A, 98, 8809-8814); (Zaremba, S, et al., 2006, Eur. J. Immunol., 36,1805-1814).

Table 1 shows the peptides, their respective SEQ ID NO: as well asinformation on the parent proteins.

TABLE 1 Peptides of the present invention SEQ ID Peptide HLA NO: CodeSequence Alleles Gene(s)  1 C20-001 ALSNLEVTL A*02 C20orf42  2 NOX-001ILAPVILYI A*02 NOX1  3 PCN-001 KLMDLDVEQL A*02 PCNA  4 PCN-002 SMSADVPLVA*02 PCNA  5 TOP-001 KIFDEILVNA A*02 TOP2A, TOP2B  6 TOP-002 AAFVEELDKVA*02 TOP2B  7 CEA-009 VLLLVHNLPQHLFG class II CEACAM5  8 TGFBI-001ALFVRLLALA A*02,A*02/ TGFBI B*13?  9 TGFBI-006 GDKLEVSLKNNVVS class IITGFBI 10 TGFBI-007 GKKLRVFVYRNSLCIENS class II TGFBI 11 TGFBI-008LKNNVVSVNKEPVAEPD class II TGFBI KNNVVSVNKEPVAEPD class II TGFBIKNNVVSVNKEPVA class II TGFBI LKNNVVSVNKEPVA class II TGFBI 12 TGFBI-009NGVIHYIDELLIPDS class II TGFBI GVIHYIDELLIPDSA class II TGFBI 13TGFBI-010 LNRILGDPEALRDL class II TGFBI 14 TGFBI-004 TPPIDAHTRNLLRNHclass II TGFBI 15 PTP-001 ALTTLMHQL A*02 PTPRZ1 16 GAL-001 SLDPSSPQVA*02 GAL3ST1 17 CHI-001 SLWAGVVVL A*02 CHI3L2 18 JAK-001 KLTDIQIEL A*02JAKMIP2 19 AKR-001 YLIHFPVSV A*02 AKR1C1, AKR1C2 20 FN1-001 IVDDITYNVA*02 FN1 21 EGFR-002 GAVRFSNNPALCNVES class II EGFR AVRFSNNPALCNVESclass II EGFR AVRFSNNPALCNVE class II EGFR 22 EGFR-005 NPTTYQMDVNPEGKYSclass II EGFR 23 EGFR-006 FKKIKVLGSGAFG class II EGFR 24 CHI3L1-001TTLIKEMKAEFIKEAQPG class 11 CHI3L1 TLIKEMKAEFIKEAQPG class II CHI3L1TTLIKEMKAEFIKEA class II CHI3L1 TLIKEMKAEFIKEA class II CHI3L1IKEMKAEFIKEAQPG class II CHI3L1 TTLIKEMKAEFIKE class II CHI3L1 25CHI3L1-007 VKSKVQYLKDRQLAG class II CHI3L1 26 CHI3L1-008 SRRTFIKSVPPFLRTclass II CHI3L1 27 DCA-001 KLGDFGLATVV A*02 DCAMKL2 28 KCN-001 SLFDQVVKVA*02 KCNJ10 29 GPM-001 ALLSEVIQL A*02 GPM6BChromosome 20 Open Reading Frame 42

C20orf42 is a focal adhesion protein involved in attachment of the actincytoskeleton to the plasma membrane and in integrin-mediated cellularprocesses. Deficiency of C20orf42 as a result of loss-of-functionmutations causes Kindler syndrome, an autosomal recessive genodermatosischaracterized by skin blistering, progressive skin atrophy,photosensitivity and, occasionally, carcinogenesis (Herz, C, et al.,2006, J Biol. Chem., 281, 36082-36090). Recently, a severegastrointestinal tract involvement with hemorrhagic colitis has beenreported in a patient with a loss-of-function mutation (Sadler, E, etal., 2006, Arch. Dermatol., 142, 1619-1624).

In the context of cancer, C20orf42 has been described within studiesinvestigating gene expression in cancer-relevant settings. It was foundto be overexpressed in 70% of colon carcinomas and 60% of lungcarcinomas tested (n=10). Normal tissue expression by Northern Blot wasrestricted to neuromuscular tissues (Weinstein, E J, et al., 2003, U,Biochim. Biophys. Acta, 1637, 207-216). Furthermore, C20orf42 has beenidentified as a gene involved in TGF-β-mediated cell migration andtumour invasion (Kloeker, S, et al., 2004, J. Biol. Chem., 279,6824-6833).

NADPH Oxidase Homolog-1 (NOX1)

NOX1, is a growth factor-responsive enzyme that catalyzes formation ofthe reactive oxygen species superoxide (O₂—) and hydrogen peroxide(H₂O₂). Its expression was originally identified in colon, prostate,uterus, and proliferating vascular smooth muscle cells (Suh, Y. A. etal. 1999; Nature 401, 79-82). Its expression is linked to a number ofbiological responses including cellular proliferation, angiogenesis, andactivation of cellular signalling pathways (Harper, R. W., et al., 2005,Arch. Biochem. Biophys. 435, 323-330).

NOX1 is highly expressed in the colon but its function in colonicphysiology or pathology is still poorly understood. In normal tissues,NOX1 expression was low in the ileum, intermediate in the right colon,and high in the left colon. There was no statistical difference in NOX1expression between samples derived from adenomas, well differentiated orpoorly differentiated colon adenocarcinomas. NOX1 was highly expressedin colon epithelial cells, both within the crypts and on the luminalsurface. In conclusion, NOX1 is an enzyme that is constitutivelyexpressed in colon epithelium and is not directly associated withtumourigenesis (Szanto, I. et al. 2005, J. Pathol. 207, 164-176).

Immunohistochemistry showed that NOX1 was constitutively expressed insurface mucous cells. Adenomas and well differentiated adenocarcinomasup-regulated NOX1 expression. Nuclear factor (NF)-kappaB waspredominantly activated in adenoma and adenocarcinoma cells expressingabundant NOX1, suggesting that NOX1 may stimulate NF-kappaB-dependentantiapoptotic pathways in colon tumours (Fukuyama, M. et al. 2005,Cancer Lett. 221, 97-104).

Wnt3a/beta-Catenin signalling has been described to induce NOX1expression (Petropoulos, H., et al., 2002, J Biol. Chem. 277,15393-15399).

Recently, reactive oxygen species have been suggested to induceendothelial apoptosis that subsequently induces the expression ofvarious adhesion molecules for tumour cells. This indicates that bytackling the production of ROS preventing tumour recurrence at distantsites might be feasible (Ten, K M, et al., 2006, Br. J Cancer, 95,1497-1503).

Proliferating Cell Nuclear Antigen (PCNA)

PCNA is found in the nucleus and is a cofactor of DNA polymerase delta.The encoded protein acts as a homotrimer and helps increase theprocessivity of leading strand synthesis during DNA replication.Therefore, it is expressed in all proliferating cells, especially tumourcells, and is used as a marker to detect proliferation.

DNA Topoisomerase II

TOP2A and TOP2B encode isoforms of a DNA topoisomerase, an enzyme thatcontrols and alters the topologic states of DNA during transcription.This nuclear enzyme is involved in processes such as chromosomecondensation, chromatid separation, and the relief of torsional stressthat occurs during DNA transcription and replication. DNA topoisomerasecatalyses the transient breaking and rejoining of two strands of duplexDNA which allows the strands to pass through one another, thus alteringthe topology of DNA. The two isoforms of this enzyme exist as likelyproducts of a gene duplication event. The gene encoding the alpha formis localised to chromosome 17 and the beta gene is localised tochromosome 3.

TOP2A is the target for several anticancer agents and a variety ofmutations in this gene have been associated with the development of drugresistance.

The TOP2A gene is located adjacent to the HER-2 oncogene, the mostfrequently amplified oncogene in breast cancer, at the chromosomelocation 17q12-q21 and is either amplified or deleted, with equalfrequency, in almost 90% of HER-2 amplified primary breast tumours(Jarvinen, T A and Liu, E T; Topoisomerase II alpha gene (TOP2A)amplification and deletion in cancer-more common than anticipated,Cytopathology, 14, 309-313). Furthermore, TOP2A amplifications have beenreported for other cancers.

Without TOP2A DNA replication and cell division are impossible. It hastherefore become the main target of many antitumour therapy regimens,even though the exact mechanism of cell killing remains elusive(Kellner, U, et al., Lancet Oncol., 3, 235-243, 2002). The success ofthis approach is limited by the development of spontaneous resistance,and drug-induced DNA damage can increase malignancy. Recent data suggestthat amplification and deletion of TOP2A may account for bothsensitivity and resistance to TOP2A-inhibitor-chemotherapy, depending onthe specific genetic defect at the TOP2A locus.

It is not clear whether the involvement of TOP2B in cancer is similar toTOP2A or whether there is a major difference between the two isoforms.TOP2B can at least supplement for some of the TOP2A activity (Sakaguchi,et al., J Cell Sci., 117, 1047-1054, 2004).

Carcinoembryonic Antigen-Related Cell Adhesion Molecule 5

Carcinoembryonic antigen (CEA=CEACAM5) is a 180 kDa heavily glycosylatedmembrane protein composed of three C21 g-like repeating units flanked bya N-terminal Ig V-like region and a C-terminal region, which includesglycophosphatidylinositol linkage region (Hegde, P, et al., Cancer Res.,61, 7792-7797, 2001).

As an oncofetal antigen, CEA is expressed during foetal development, butalso, at low levels, in the gastrointestinal epithelium of adults.However, CEA is overexpressed in a high percentage of human tumours,including 90% of gastrointestinal, colorectal and pancreatic cancer, 70%of non-small cell lung cancer cells and 50% of breast cancers (Thompson,J A, et al., J Clin Lab Anal., 5, 344-366, 2005). Due to its highexpression by tumour cells and its secretion to the serum, CEA has beenbroadly used as a tumour marker (Sikorska, H, et al., Cancer Detect.Prev., 12, 321-355, 1988) and is the standard serum marker forcolorectal cancer monitoring (Locker, G Y, et al., J Clin Oncol, 24,5313-5327, 2006).

Despite the overexpression of CEA in tumour cells, cancer patients donot normally show an immune response against this antigen (Orefice, S,et al., Tumouri, 68, 473-475, 1982) The immune system commonly becomestolerant to CEA, because it is normally expressed at low levels in thebody. However, in a series of clinical vaccine trials, theimmunogenicity of CEA has been demonstrated (Sarobe, P, et al., Curr.Cancer Drug Targets., 4, 443-454, 2004), especially in colorectalcarcinoma (CRC) (Mosolits, S, et al., Ann. Oncol., 16, 847-862, 2005),and CEA is the tumour associated antigen (TAA) with the greatest numberof vaccine platforms tested in this tumour type (von Mehren, M; Oncol.,32, 76-84, 2005).

Several cytotoxic and helper T-cell epitopes have been described for CEA(Crosti, M, et al., J. Immunol., 176, 5093-5099, 2006; Novellino, L, etal., Cancer Immunol. Immunother., 54, 187-207, 2005; Ruiz, M, et al.,Clin Cancer Res., 10, 2860-2867, 2004), enabling a variety ofpeptide-based vaccination trials in CRC (Babatz, J, et al, CancerImmunol. Immunother., 55, 268-276, 2006; Fong, L, et al., Proc. Natl.Acad. Sci. U.S.A, 98, 8809-8814, 2001; Liu, K J, et al., Clin CancerRes., 10, 2645-2651, 2004; Matsuda, K, et al., Cancer Immunol.Immunother., 53, 609-616, 2004; Ueda, Y, et al., Int. J. Oncol., 24,909-917, 2004; Weihrauch, M R, et al., Clin Cancer Res., 11, 5993-6001,2005). These and other clinical trials to date have demonstrated safetyof CEA vaccinations and evidence for the induction of immune responseagainst this antigen (von Mehren, M; Semin. Oncol., 32, 76-84, 2005).

Transforming Growth Factor, Beta-Induced (TGFBI)

TGFBI was first identified as a TGF-beta-inducible gene in a human lungadenocarcinoma cell line. It encodes for a secreted extracellular matrixprotein, which is thought to act on cell attachment and extracellularmatrix composition.

TGFBI was shown to be among the most significantly elevated genes incolorectal cancers and it is expressed at high levels in adenomas aswell. Quantitative PCR results demonstrated strong elevation in bothunpurified tumours and purified tumour epithelial cells. Accordingly, insitu hybridization experiments revealed TGFBI to be expressed in manycell types, in both the stromal and epithelial compartments (Buckhaults,P, et al., Cancer Res., 61, 6996-7001, 2001).

In a meta-analysis of studies investigating gene expression incolorectal carcinoma, TGFBI was identified as one of only nine genesdescribed as upregulated repeatedly (4 studies for TGFBI) (Shih, W, etal., Oncol. Rep., 13, 517-524, 2005).

In human pancreatic tissues, there was a 32.4-fold increase in TGFBImRNA levels in pancreatic cancers in comparison to normal controltissues. In situ hybridization analysis revealed that TGFBI mRNA wasexpressed mainly in the cancer cells within the pancreatic tumour mass(Schneider, D, et al., Biochim. Biophys. Acta, 1588, 1-6, 2002).

TGFBI was identified as a gene promoting angiogenesis in an in vitromodel. Additionally, dramatically enhanced expression of TGFBI wasdetected in several tumours. Antisense oligonucleotides to TGFBI blockedboth gene expression and endothelial tube formation in vitro, suggestingthat TGFBI may play a critical role in endothelial cell-matrixinteractions (Aitkenhead, M, et al., Microvasc. Res., 63, 159-171,2002).

Protein Tyrosine Phosphatase, Receptor-Type, Zeta1 (PTPRZ1)

PTPRZ1 is a member of the receptor type protein tyrosine phosphatasefamily and encodes a single-pass type I membrane protein with twocytoplasmic tyrosine-protein phosphatase domains, an alpha-carbonicanhydrase domain and a fibronectin type-III domain. Expression of thisgene is induced in gastric cancer cells (Wu, C W, et al., Cancer Lett.,242, 95-103, 2006), in the remyelinating oligodendrocytes of multiplesclerosis lesions (Harroch, S, et al., Nat. Genet., 32, 411-414, 2002),and in human embryonic kidney cells under hypoxic conditions (Wang, V,et al., Cancer Res., 65, 3299-3306, 2005).

Both the protein and transcript are overexpressed in glioblastoma cells,promoting their haptotactic migration (Lu, K V, et al., J Biol. Chem.,280, 26953-26964, 2005).

Furthermore, PTRPZ1 is frequently amplified at the genomic DNA level inglioblastoma (Mulholland, P J, et al., Cell Cycle, 5, 783-791, 2006).

Janus Kinase and Microtubule Interacting Protein 2 (JAKMIP2)

JAKMIP2 was identified as one of many known or putative downstreamtargets of PAX3-FKHR which were highly overexpressed in ARMS (Paediatricrhabdomyosarcoma, alveolar subtype) (Lae, M, et al., 2007, J. Pathol.,212, 143-151).

Fibronectin 1 (FN1)

Fibronectin is a high-molecular-weight glycoprotein containing about 5%carbohydrate that binds to receptor proteins that span the cell'smembrane, called integrins. In addition to integrins, they also bindextracellular matrix components such as collagen, fibrin and heparin.There are several isoforms of fibronectin, all of which are the productof a single gene. FNs play a critical role in the maintenance of normalcell morphology, cell adhesion, migration, hemostasis, thrombosis, woundhealing, differentiation and proliferation (Hynes, R O, Sci. Am., 254,42-51, 1987).

The polymeric fibronectin, sFN, is formed in vitro by treating solublefibronectin with a 76-aa peptide, III1-C (called Anastellin), which isderived from the first type III repeat in fibronectin. In vivo studiesin tumour-bearing mice showed that systemic application of Anastellin orsFN suppressed tumour growth, angiogenesis and metastasis (Yi, M et al.,Proc. Natl. Acad. Sci. U.S.A, 98, 620-624, 2001). Anginex is a synthetic33-amino acid peptide that was originally modelled to reproduce thebeta-sheet structure of antiangiogenic proteins. It has been shown thatanginex initiates fibronectin polymerization and is inactive in micethat lack plasma fibronectin (Akerman, M E, et al., Proc. Natl. Acad.Sci. U.S.A, 102, 2040-2045). In a study, they examined the effects of FNon D-galactosamine (GalN)/lipopolysaccharide (LPS)-induced fulminantliver failure in mice. The results suggest that FN protected againstGalN/LPS-induced liver failure by a mechanism involving inhibition ofNF-kappaB activation, which caused down-regulation of TNF-alpha andinvolved up-regulation of IL-10, and elevation of Bcl-xL induced ablockage of apoptotic signals, by which apoptosis of hepatocytes causedby Ga1N/LPS was suppressed (Qiu, Z, et al., Shock, 25, 80-87, 2006).Other results indicate that FN stimulates human lung carcinoma cellproliferation and diminishes apoptosis in vitro by inducing COX-2 geneexpression and PGE2 biosynthesis (Han, S., et al., Int. J. Cancer, 111,322-331, 2004).

Fibronectin (FN) has been shown to undergo alternative splicingexclusively during organogenesis and tumourigenesis. One such splicevariant, extradomain-B (ED-B) FN, is normally absent in normal adulttissues and is proposed to be a marker of tumoural angiogenesis (Khan, ZA, et al., Exp. Lung Res., 31, 701-711, 2005). Mhawech et al. showedthat head and neck tumours with a positive staining for EDB had a trendto a significant lower overall survival of patients (Mhawech, P, et al.,Oral Oncol., 41, 82-88, 2005).

Fibronectin expression regulates angiogenesis and vasculogenesis andparticipates in brain tissue responses to ischemia and seizures. Thegene expression of fibronectin was significantly increased (p<0.05) inthe SWS (Sturge-Weber syndrome) fibroblasts compared with that offibroblasts from SWS normal skin (Comi, A M, et al., Pediatr. Res., 53,762-7692, 2003). The fibronectin concentration was significantly higherin ovarian cancers compared with benign ovarian tumours and normalovaries. Fibronectin concentration significantly elevated in ovariancancer patients with recurrent disease compared with ovarian cancerpatients without recurrence. The expression of tumour-derivedmatriolytic enzymes and fibronectin are important in the growth ofovarian tumours (Demeter, A, et al., Orv. Hetil., 145, 1617-1624, 2004).The fact that FN was one of the only two genes significantlydown-regulated out of the 1,176 genes analyzed in a study stresses thehypothesis that FN may behave as an important metastasis suppressor genein mammary cancer (Urtreger, A J, et al., Oncol. Rep., 16, 1403-1410).

In a report, they found that three soluble fibronectin peptides (RGD,CS-1, and FN-C/H-V) induce apoptosis in lung fibroblasts. Apoptosisoccurred by disruption of adhesion (anoikis). The use of smallfibronectin peptides to promote fibroblast apoptosis warrants furtherstudy as possible antifibrotic therapy (Hadden, H L et al., Am. J.Respir. Crit. Care Med, 162, 1553-1560, 2000). Another study hasdemonstrated that fibronectin (FN) stimulates human non-small cell lungcarcinoma (NSCLC) cell proliferation. They show that FN increases MMP-9protein, mRNA expression, and gelatinolytic activity in NSCLC cells(Han, S, et al., J Biol. Chem., 281, 29614-29624, 2006). In one study,they investigated whether the tumour-suppressive effects of vitamin D(VD) compounds may also be mediated by mechanisms that govern celladhesiveness. Introduction of small interfering RNA against FN resultedin down-regulation of FN expression and diminished cell adhesiveness toa collagen-type I matrix. Their findings highlight the significance ofFN in modulating thyroid cancer cell adhesiveness and, at least in part,in mediating VD actions on neoplastic cell growth (Liu, W, et al. Mol.Endocrinol., 19, 2349-2357, 2005).

The generation of tumour-associated FN isoforms allows the developmentof specific ligands (e.g., antibodies), which can be used for theselective delivery of therapeutic agents to the tumour environment. FNis being used as a target for biomolecular intervention, both for thedevelopment of inhibitory molecules that block the interaction of FNwith integrins and other receptors on the cell surface, and for thedevelopment of ligand-based targeted imaging and therapeutic strategies(Kaspar, M, et al., Int. J. Cancer, 118, 1331-1339, 2005). One studydemonstrated that the treatment by in vivo expression of a recombinantCBD-HepII polypeptide of FN, designated as CHSO, strongly inhibited thetumour growth, tumour invasion and angiogenesis. The gene therapy withCHSO not only prolonged the survival of mice bearing hepatocarcinoma inthe liver, but also suppressed the growth and invasive ability of tumourin spleen and its metastasis to liver. Taken together, these findingssuggest a prospective utility of CHSO in the gene therapy of livercancer (Liu, Y, et al., Int. J. Cancer, 2007, 121(1):184-92).Fibronectin (FN) has a cryptic functional site (YTIYVIAL sequence withinthe 14th type III repeat) opposing cell adhesion to extracellularmatrix. A 22-mer FN peptide containing this site, termed FNIII14,inhibits beta1 integrin-mediated adhesion without binding to integrins.The study shows that FNIII14 has the potential to prevent lymphoma cellmetastasis (Kato, R, et al., Clin Cancer Res., 8, 2455-2462, 2002).

Epidermal Growth Factor Receptor (EGFR)

EGFR plays an important role in the regulation of normal cellproliferation, differentiation and survival. For this reason EGFR statusis often altered in a range of human tumour types and generallycorrelates with a poor prognosis. In neoplastic cells it contributes totheir growth and survival through various divergent pathways (Maehama,T, et al., J Biol. Chem., 273, 13375-13378, 1998). EGFR abnormalitiesare one of the most common molecular aberrations in glioblastoma(Zawrocki, A et al., Folia Neuropathol., 43, 123-132, 2005).

The EGFR amplification and mRNA overexpression are frequent in highgrade gliomas of astrocytic origin, and are always strongly associatedwith an increased level of the EGFR protein (Wong, A J, et al., 1987,Proc. Natl. Acad. Sci. U.S.A, 84, 6899-6903; Chaffanet, M, et al., 1992,Eur. J. Cancer, 28, 11-17). Protein overexpression without geneamplification has been reported in up to 27% of GBMs, but less malignantastrocytomas and oligodendrogliomas were also reported to demonstratethe EGFR overexpression without the underlying gene amplification(Reifenberger, J, et al., 1996, Am. J. Pathol., 149, 29-35).

The prognostic implications of the EGFR amplification/overexpression inbrain tumours are controversial. Some authors did not find any influenceof the EGFR amplification/overexpression on survival of the patients(Olson, J J, et al., 1998, Clin Cancer Res., 4, 215-222; Newcomb, E W,et al., 1998, Brain Pathol., 8, 655-667; Waha, A, et al., 1996, JNeurosurg., 85, 634-641) while the others concluded that thesealterations were a negative prognostic factor (Etienne, M C, et al.,1998, Clin Cancer Res., 4, 2383-2390; Jaros, E, et al., 1992, Br. J.Cancer, 66, 373-385; Schlegel, J, et al., 1994, Int. J. Cancer, 56,72-77; Zhu, A, et al., 1996, Int. J. Radiat. Oncol. Biol Phys., 34,809-815).

There exist a few treatment approaches to the EGFR molecule on thecancer cell. The most extensively studied include: specific antibodytherapy by means of unarmed antibodies or antibodies conjugated withtoxins, liposomes or nuclides, and the use of inhibitors of the receptortyrosine kinase. There are several types of monoclonal antibodiesdirected against the EGFRwt. Their use results in blocking access to thereceptor for its ligands (cetuximab) and/or rapid internalization of thereceptor (ABX-EGF) (Sridhar, S S, et al., 2003, Lancet Oncol., 4,397-406). As the EGFRwt occurs also on the surface of normal cells, sideeffects may limit its use.

EGFR is overexpressed in head and neck squamous cell carcinoma (HNSCC)where expression levels correlate with decreased survival. Therapiesthat block EGFR have shown limited efficacy in clinical trials andprimarily when combined with standard therapy. EGFRvIII is expressed inHNSCC where it contributes to enhanced growth and resistance totargeting wild-type EGFR. The antitumour efficacy of EGFR targetingstrategies may be enhanced by the addition of EGFRvIII-specific blockade(Sok, J C, et al., 2006, Clin Cancer Res., 12, 5064-5073).

Another strategy is to selectively induce the death of glioblastomacells and other cancer cells that over-express the EGF receptor. Using anon-viral delivery vector that homes to the EGF receptor, syntheticanti-proliferative dsRNA (polyinosine-cytosine [poly IC]), a strongactivator of apoptosis, was targeted selectively to cancer cells.EGFR-targeted poly IC induced rapid apoptosis in the target cells invitro and in vivo. Tumoural delivery of the EGFR-targeted poly ICinduced the complete regression of pre-established intracranial tumoursin nude mice, with no obvious adverse toxic effects on normal braintissue. A year after treatment completion the treated mice remaincancer-free and healthy (Shir, A, et al., 2006, PLoS. Med, 3, e6-).

The application of small interfering RNAs (siRNAs) has become aneffective and highly specific tool to modulate gene expression, and awide range of oncogenes have been silenced successfully. siRNA-mediateddown-regulation of EGFR was shown in two established glioma cell lineswith different EGFR expression levels (U373 MG, LN18). The expression ofEGFR mRNA and protein was down-regulated by 70-90%. However, siRNAtreatment had no inhibitory effect on cell proliferation, migration andactivation status of EGFR-coupled signalling cascades. In accordancewith these results, gene expression analysis with microarrays revealedonly small, albeit specific changes in expression patterns. Inconclusion, these data indicate that the specific down-regulation ofEGFR might not be sufficient for a single agent therapeutic approach inmalignant glioma (Vollmann, A, et al., 2006, Int. J. Oncol., 28,1531-1542).

Several clinical studies exist have been conducted that show promisingresults. For example, h-R3 is a humanized monoclonal antibody thatrecognize the EGFR external domain with high affinity, inhibitingtyrosine kinase activation. To evaluate safety, immunogenicity andpreliminary efficacy of h-R3 in newly diagnosed high-grade gliomapatients, a Phase I/II trial was conducted (Ramos, T C, et al., 2006,Cancer Biol Ther., 5, 375-379).

EKB-569 is a potent, low molecular weight, selective, and irreversibleinhibitor of epidermal growth factor receptor (EGFR) that is beingdeveloped as an anticancer agent. A phase 1, dose-escalation study wasconducted in Japanese patients. Based on RECIST criteria, they hadstable disease but radiographic tumour regression was observed(Yoshimura, N, et al., 2006, Lung Cancer, 51, 363-368).

Gefitinib, a specific inhibitor of epidermal growth factor receptor(EGFR)-associated tyrosine kinase has demonstrated efficacy in asubgroup of patients with non-small-cell lung carcinoma (NSCLC) who failconventional chemotherapy. It is also reported to have an antitumoureffect in brain metastases from NSCLC. Additionally, EGFR mutations haveshown a strong association with gefitinib sensitivity for NSCLC. Theefficacy of gefitinib in brain metastases from NSCLC was assessed andthe association of this efficacy with EGFR mutations evaluated.Gefitinib appears to be effective in treating brain metastases in asubgroup of patients. The data suggested a possible association betweenthe efficacy of gefitinib in the treatment of brain metastases and EGFRmutations (Shimato, S, et al., 2006, Neuro.-oncol., 8, 137-144).

Chitinase 3-Like 2 (CHI3L2)

CHI3L2 was originally identified from chondrocytes. It has beenfrequently described as a target antigen in rheumatoid arthritis. Norelevant association of CHI3L2 with cancer was identified. Chitinase3-like proteins have been implied in stimulating proliferation of humanconnective tissue cells, e.g. fibroblasts, by activating extracellularsignal-regulated kinase and PKB mediated signalling pathways (Recklies AD, et al., Biochem J. 2002; 365:119-126). In mice chitinase 3-likeproteins have been found to be strongly upregulated inHelicobacter-induced gastric cancer models (Takaishi S, et al. CancerSci. 2007 (3): 284-293)

Doublecortin and CaM Kinase-Like 2 (DCAMKL2)

The microtubule (MT)-associated DCX protein plays an essential role inthe development of the mammalian cerebral cortex. Identification of aprotein kinase, doublecortin kinase-2 (DCAMKL2), with a domain (DC)highly homologous to DCX was reported. DCAMKL2 has MT binding activityassociated with its DC domain and protein kinase activity mediated by akinase domain, organized in a structure in which the two domains arefunctionally independent.

Overexpression of DCAMKL2 stabilizes the MT cytoskeleton againstcold-induced depolymerization. Autophosphorylation of DCAMKL2 stronglyreduces its affinity for MTs. DCAMKL2 and DCX mRNAs are nervoussystem-specific and are expressed during the period of cerebrocorticallamination. DCX is down-regulated postnatally, whereas DCAMKL2 persistsin abundance into adulthood, suggesting that the DC sequence haspreviously unrecognized functions in the mature nervous system. Insympathetic neurons, DCAMKL2 is localized to the cell body and to theterminal segments of axons and dendrites.

DCAMKL2 may represent a phosphorylation-dependent switch for thereversible control of MT dynamics in the vicinity of neuronal growthcones. The patterns of expression, functional activities, regulation,and localization of DCAMKL2 suggest that it functions in parallel to, orin concert with, other members of the DC gene family (DC domain-encodinggenes) in events important for neural development and, potentially, inthose characteristic of mature nervous systems. DCAMKL2 is composed oftwo functional and independent domains, an MT-binding and -stabilizingdomain (the DC sequence) and a kinase domain with proteinphosphotransferase activity.

It was suggested that the DC sequence plays a critical role intransducing extracellular cues and their intracellular signals intochanges in MT dynamics. In particular, based on an ability to interactwith MTs in a fashion regulated by phosphorylation and to localize toterminal segments of axons and dendrites, regions in which MTs aredynamically unstable, DCAMKL2 should be considered a potential candidatemediator of the rapid cytoskeletal rearrangements that occur in responseto neuronal signalling events (Edelman, A M, et al., 2005, J Biol.Chem., 280, 8531-8543).

ATP-Sensitive Inward Rectifier Potassium Channel 10 (KCNJ10)

The major function of inwardly rectifying potassium channels (Kir) is inestablishing the high potassium (K+) selectivity of the glial cellmembrane and strongly negative resting membrane potential (RMP), whichare characteristic physiological properties of glia. The classicalproperty of Kir is that K+ flows inwards when the RMP is negative to theequilibrium potential for K+(E(K)), but at more positive potentialsoutward currents are inhibited. A feature of CNS glia is their specificexpression of the KCNJ10 subtype, which is a major K+ conductance inglial cell membranes and has a key role in setting the glial RMP. Hence,Kir, and in particular KCNJ10 are key regulators of glial functions,which in turn determine neuronal excitability and axonal conduction(Butt, A M et al., 2006, J Cell Mol. Med, 10, 33-44).

Diminished potassium and glutamate buffering capabilities of astrocytesresult in hyperexcitability of neurons and abnormal synaptictransmission. KCNJ10 channels are primarily responsible for significanthyperpolarization of cortical astrocytes and are likely to play a majorrole in potassium buffering. Significant inhibition of glutamateclearance in astrocytes with knock-down of KCNJ10 highlights the role ofmembrane hyperpolarization in this process (Kucheryavykh, Y V, et al.,2006, Glia, Volume 55 Issue 3, Pages 274-281).

KCNJ10 spatial buffering of extracellular K(+) in the central nervoussystem can only be performed due to the non-uniform distribution ofKCNJ10 across the surface of the glial cell. A mislocalization of KCNJ10in various human brain tumours (low- and high-grade astrocytomas andoligodendrogliomas) was observed, suggesting that buffering capacity ofglial cells may be compromised, leading to water influx (cytotoxicedema) (Warth, A, et al., 2005, Acta Neuropathol. (Berl), 109, 418-426).KCNJ10 was also upregulated in astrocytes in damaged brain. Thefollowing hypothesis was proposed: in astrocytes, under normalconditions, AQP4 couples water transport with KCNJ10 mediated K+siphoning, but in pathological states, AQP4 facilitates the flow ofbrain oedema fluid, and KCNJ10 buffers increased extracellular K+(Saadoun, S, et al., 2003, J Clin Pathol., 56, 972-975).

In addition to the peptides shown in SEQ ID NO:1-29, as discussed above,the present invention further includes variants of these peptides aslong as they will induce T-cells cross reacting with the peptide. By a“variant” of the given amino acid sequence the inventors mean that theside chains of, for example, one or two of the amino acid residues arealtered (for example by replacing them with the side chain of anothernaturally occurring amino acid residue or some other side chain) so thatthe peptide is still able to bind to an HLA molecule in substantiallythe same way as a peptide consisting of the given amino acid sequence.For example, a peptide may be modified so that it at least maintains, ifnot improves, the ability to interact with and bind a suitable MHCmolecule, such as HLA-A or -DR, and so that it at least maintains, ifnot improves, the ability to generate activated CTL that can recogniseand kill cells that express a polypeptide containing an amino acidsequence as defined in the aspects of the invention. As can derived fromthe database, certain positions of HLA-A binding peptides are typicallyanchor residues forming a core sequence fitting to the binding motif ofthe HLA binding groove.

Those amino acid residues that are not essential to interact with theT-cell receptor can be modified by replacement with another amino acidwhose incorporation does not substantially effect T-cell reactivity anddoes not eliminate binding to the relevant MHC. Thus, apart from theproviso given, the peptide of the invention may be any peptide (by whichterm the inventors include oligopeptide or polypeptide) which includesthe amino acid sequences or a portion or variant thereof as given.

It is furthermore known for MHC-class II presented peptides that thesepeptides are composed of a “core sequence” having a certain HLA-specificamino acid motif and, optionally, N- and/or C-terminal extensions thatdo not interfere with the function of the core sequence (i.e. are deemedas irrelevant for the interaction of the peptide and all or a subset ofT-cell clones recognising the natural counterpart). The N- and/orC-terminal extensions can, for example, be between 1 to 10 amino acidsin length, respectively. These peptides can be used either directly toload MHC class II molecules or the sequence can be cloned into thevectors according to the description herein below. As these peptidesconstitute the final product of the processing of larger peptides withinthe cell, longer peptides can be used as well. The peptides of theinvention may be of any size, but typically they may be less than100,000 in molecular weight, preferably less than 50,000, morepreferably less than 10,000 and typically about 5,000. In terms of thenumber of amino acid residues, the peptides of the invention may havefewer than 1000 residues, preferably fewer than 500 residues, morepreferably fewer than 100 residues. Accordingly the present inventionalso provides peptides and variants thereof wherein the peptide orvariant has an overall length of between 8 and 100, preferably between 8and 30, and most preferred between 8 and 16, namely 8, 9, 10, 11, 12,13, 14, 15, or 16 amino acids.

Correspondingly, naturally occurring or artificial variants that induceT-cells cross-reacting with a peptide of the invention are often lengthvariants. Examples for such naturally occurring length variants aregiven in Table 1 for SEQ ID NOs: 11 and 12, and 21 and 24, respectively.

If a peptide longer than around 12 amino acid residues is used directlyto bind to a MHC class II molecule, it is preferred that the residuesthat flank the core HLA binding region do not substantially affect theability of the peptide to bind specifically to the binding groove of theMHC class II molecule or to present the peptide to the CTL. However, asalready indicated above, it will be appreciated that larger peptides maybe used, e.g. when encoded by a polynucleotide, since these largerpeptides may be fragmented by suitable antigen-presenting cells.

It is also possible, that MHC class I epitopes, although usually between8-10 amino acids long, are generated by peptide processing from longerpeptides or proteins that include the actual epitope. Similar to MHCclass II epitopes, it is preferred that the residues that flank thebinding region do not substantially affect the ability of the peptide tobind specifically to the binding groove of the MHC class 1 molecule orto present the peptide to the CTL nor mask the sites for proteolyticcleavage necessary to expose the actual epitope during processing.

Accordingly the present invention also provides peptides and variants ofMHC class I epitopes having an overall length of between 8 and 100,preferably between 8 and 30, and most preferred between 8 and 16, namely8, 9, 10, 11, 12, 13, 14, 15, or 16 amino acids.

Of course, the peptide or variant according to the present inventionwill have the ability to bind to a molecule of the human majorhistocompatibility complex (MHC) class I or II. Binding of a peptide ora variant to a MHC complex may be tested by methods known in the art,for example those described in example 4 of the present invention orthose described in the literature for different MHC class II alleles(e.g. Vogt A B, et al., J. Immunol. 1994; 153(4):1665-1673; Malcherek G,et al., J. Immunol. 1994; 153(3):1141-1149; Manici S, et al., J Exp Med.1999; 189(5): 871-876; Hammer J, et al., J Exp Med. 1995181(5):1847-1855; Tompkins S M, et al., J Immunol Methods. 1993; 163(2):209-216; Boyton R J, et al., Int Immunol. 1998 (12):1765-1776).

In a particularly preferred embodiment of the invention, the peptideconsists or consists essentially of an amino acid sequence according toSEQ ID NO: 1 to SEQ ID NO: 29.

“Consisting essentially of” shall mean that a peptide according to thepresent invention, in addition to the sequence according to any of SEQID NO: 1 to SEQ ID NO: 29 or a variant thereof, contains additional N-and/or C-terminally located stretches of amino acids that are notnecessarily forming part of the peptide that functions as an epitope forMHC molecules epitope.

Nevertheless, these stretches can be important to provide an efficientintroduction of the peptide into the cells. In one embodiment of thepresent invention, the peptide of the present invention is a fusionprotein which comprises, for example, the 80 N-terminal amino acids ofthe HLA-DR antigen-associated invariant chain (p33, in the following“Ii”) as derived from the NCBI, GenBank Accession-number X00497(Strubin, M., at al., EMBO J. 3 (4), 869-872 (1984)).

In addition the peptide or variant may be modified further to improvestability and/or binding to MHC molecules to elicit a stronger immuneresponse. Methods for such an optimization of a peptide sequence arewell known in the art and include, for example, the introduction ofreverse peptide bonds or non-peptide bonds.

In a reverse peptide bond amino acid residues are not joined by peptide(—CO—NH—) linkages but the peptide bond is reversed. Such retro-inversopeptidomimetics may be made using methods known in the art, for examplesuch as those described in Meziere et al (1997) J. Immunol. 159,3230-3237, incorporated herein by reference. This approach involvesmaking pseudopeptides containing changes involving the backbone, and notthe orientation of side chains. Meziere et al (1997) show that for MHCand T helper cell responses, these pseudopeptides are useful.Retro-inverse peptides containing NH—CO bonds instead of CO—NH peptidebonds, are much more resistant to proteolysis.

A non-peptide bond is, for example, —CH₂—NH, —CH₂S—, —CH₂CH₂—, —CH═CH—,—COCH₂—, —CH(OH)CH₂—, and —CH₂SO—. U.S. Pat. No. 4,897,445 provides amethod for the solid phase synthesis of non-peptide bonds (—CH₂—NH) inpolypeptide chains that involves polypeptides synthesised by standardprocedures and the non-peptide bond synthesised by reacting an aminoaldehyde and an amino acid in the presence of NaCNBH₃.

Peptides comprising the sequences described above may be synthesizedwith additional chemical groups present at their amino and/or carboxytermini, to enhance, for example, the stability, bioavailability, and/oraffinity of the peptides. For example, hydrophobic groups such ascarbobenzoxyl, dansyl, or t-butyloxycarbonyl groups may be added to thepeptides' amino termini. Likewise, an acetyl group or a9-fluorenylmethoxy-carbonyl group may be placed at the peptides' aminotermini. Additionally, the hydrophobic group, t-butyloxycarbonyl, or anamido group may be added to the peptides' carboxyl termini.

Further, the peptides of the invention may be synthesized to alter theirsteric configuration. For example, the D-isomer of one or more of theamino acid residues of the peptide may be used, rather than the usualL-isomer. Still further, at least one of the amino acid residues of thepeptides of the invention may be substituted by one of the well knownnon-naturally occurring amino acid residues. Alterations such as thesemay serve to increase the stability, bioavailability and/or bindingaction of the peptides of the invention.

Similarly, a peptide or variant of the invention may be modifiedchemically by reacting specific amino acids either before or aftersynthesis of the peptide. Examples of these modifications are well knownin the art and are summarised in for example, R. Lundblad, ChemicalReagents for Protein Modification, 3rd ed. CRC Press, 2005, which isincorporated herein by reference. Chemical modification of amino acidsincludes, but is not limited to, modification by acylation, amidination,pyridoxylation of lysine, reductive alkylation, trinitrobenzylation ofamino groups with 2,4,6-trinitrobenzene sulphonic acid (TNBS), amidemodification of carboxyl groups and sulphydryl modification by performicacid oxidation of cysteine to cysteic acid, formation of mercurialderivatives, formation of mixed disulphides with other thiol compounds,reaction with maleimide, carboxymethylation with iodoacetic acid oriodoacetamide and carbamoylation with cyanate at alkaline pH, althoughwithout limitation thereto. In this regard, the skilled person isreferred to Chapter 15 of Current Protocols In Protein Science, Eds.Coligan et al. (John Wiley & Sons NY, 1995-2000) for more extensivemethodology relating to chemical modification of proteins.

Briefly, modification of e.g. arginyl residues in proteins is oftenbased on the reaction of vicinal dicarbonyl compounds such asphenylglyoxal, 2,3-butanedione, and 1,2-cyclohexanedione to form anadduct. Another example is the reaction of methylglyoxal with arginineresidues. Cysteine can be modified without concomitant modification ofother nucleophilic sites such as lysine and histidine. As a result, alarge number of reagents are available for the modification of cysteine.The websites of Pierce Chemical Company and Sigma-Aldrich and othersprovide information on specific reagents.

Selective reduction of disulfide bonds in proteins is also common.Disulfide bonds can be formed and oxidized during the heat treatment ofbiopharmaceuticals.

Woodward's Reagent K may be used to modify specific glutamic acidresidues. N-(3-(dimethylamino)propyl)-N′-ethylcarbodiimide can be usedto form intra-molecular crosslinks between a lysine residue and aglutamic acid residue. For example, diethylpyrocarbonate is a reagentfor the modification of histidyl residues in proteins. Histidine canalso be modified using 4-hydroxy-2-nonenal. The reaction of lysineresidues and other α-amino groups is, for example, useful in binding ofpeptides to surfaces or the cross-linking of proteins/peptides. Lysineis the site of attachment of poly(ethylene)glycol and the major site ofmodification in the glycation of proteins. Methionine residues inproteins can be modified with e.g. iodoacetamide, bromoethylamine,chloramine T. Tetranitromethane and N-acetylimidazole can be used forthe modification of tyrosyl residues. Cross-linking via the formation ofdityrosine can be accomplished with hydrogen peroxide/copper ions.Recent studies on the modification of tryptophan have usedN-bromosuccinimide, 2-hydroxy-5-nitrobenzyl bromide or3-bromo-3-methyl-2-(2-nitrophenylmercapto)-3H-indole (BPNS-skatole).

Successful modification of therapeutic proteins and peptides with PEG isoften associated with an extension of circulatory half-life whilecross-linking of proteins with glutaraldehyde, polyethyleneglycoldiacrylate and formaldehyde is used for the preparation of hydrogels.Chemical modification of allergens for immunotherapy is often achievedby carbamylation with potassium cyanate.

A peptide or variant, wherein the peptide is modified or includesnon-peptide bonds is a preferred embodiment of the invention.

Generally, peptides and variants (at least those containing peptidelinkages between amino acid residues) may be synthesised by theFmoc-polyamide mode of solid-phase peptide synthesis as disclosed by Luet al (1981) J. Org. Chem. 46, 3433 and references therein. TemporaryN-amino group protection is afforded by the 9-fluorenylmethyloxycarbonyl(Fmoc) group. Repetitive cleavage of this highly base-labile protectinggroup is done using 20% piperidine in N, N-dimethylformamide. Side-chainfunctionalities may be protected as their butyl ethers (in the case ofserine threonine and tyrosine), butyl esters (in the case of glutamicacid and aspartic acid), butyloxycarbonyl derivative (in the case oflysine and histidine), trityl derivative (in the case of cysteine) and4-methoxy-2,3,6-trimethylbenzenesulphonyl derivative (in the case ofarginine). Where glutamine or asparagine are C-terminal residues, use ismade of the 4,4′-dimethoxybenzhydryl group for protection of the sidechain amido functionalities. The solid-phase support is based on apolydimethyl-acrylamide polymer constituted from the three monomersdimethylacrylamide (backbone-monomer), bisacryloylethylene diamine(cross linker) and acryloylsarcosine methyl ester (functionalisingagent). The peptide-to-resin cleavable linked agent used is theacid-labile 4-hydroxymethyl-phenoxyacetic acid derivative. All aminoacid derivatives are added as their preformed symmetrical anhydridederivatives with the exception of asparagine and glutamine, which areadded using a reversedN,N-dicyclohexyl-carbodiimide/1hydroxybenzotriazole mediated couplingprocedure. All coupling and deprotection reactions are monitored usingninhydrin, trinitrobenzene sulphonic acid or isotin test procedures.Upon completion of synthesis, peptides are cleaved from the resinsupport with concomitant removal of side-chain protecting groups bytreatment with 95% trifluoroacetic acid containing a 50% scavenger mix.Scavengers commonly used are ethandithiol, phenol, anisole and water,the exact choice depending on the constituent amino acids of the peptidebeing synthesized. In addition, a combination of solid phase andsolution phase methodologies for the synthesis of peptides is possible(see, for example, Bruckdorfer T, et al., Curr Pharm Biotechnol. 2004February; 5(1):29-43 and the references as cited therein).Trifluoroacetic acid is removed by evaporation in vacuo, with subsequenttrituration with diethyl ether affording the crude peptide. Anyscavengers present are removed by a simple extraction procedure which onlyophilization of the aqueous phase affords the crude peptide free ofscavengers. Reagents for peptide synthesis are generally available frome.g. Calbiochem-Novabiochem (UK) Ltd, Nottingham NG7 2QJ, UK.

Purification may be effected by any one, or a combination of, techniquessuch as re-crystallization, size exclusion chromatography, ion-exchangechromatography, hydrophobic interaction chromatography and (usually)reverse-phase high performance liquid chromatography using e.g.acetonitril/water gradient separation.

Analysis of peptides may be carried out using thin layer chromatography,electrophoresis, in particular capillary electrophoresis, solid phaseextraction (CSPE), reverse-phase high performance liquid chromatography,amino-acid analysis after acid hydrolysis and by fast atom bombardment(FAB) mass spectrometric analysis, as well as MALDI and ESI-Q-TOF massspectrometric analysis.

A further aspect of the invention provides a nucleic acid (e.g.polynucleotide) encoding a peptide or peptide variant of the invention.The polynucleotide may be e.g. DNA, cDNA, PNA, CNA, RNA, mRNA, and siRNAor combinations thereof, either single- and/or double-stranded, ornative or stabilised forms of polynucleotides, such as e.g.polynucleotides with a phosphorothiate backbone, and it may or may notcontain introns so long as it codes for the peptide. Of course, onlypeptides containing naturally occurring amino acid residues joined bynaturally occurring peptide bonds are encodable by a polynucleotide. Astill further aspect of the invention provides an expression vectorcapable of expressing a polypeptide according to the invention.

A variety of methods have been developed to operably linkpolynucleotides, especially DNA, to vectors for example viacomplementary cohesive termini. For instance, complementary homopolymertracts can be added to the DNA segment to be inserted to the vector DNA.The vector and DNA segment are then joined by hydrogen bonding betweenthe complementary homopolymeric tails to form recombinant DNA molecules.

Synthetic linkers containing one or more restriction sites provide analternative method of joining the DNA segment to vectors. Syntheticlinkers containing a variety of restriction endonuclease sites arecommercially available from a number of sources including InternationalBiotechnologies Inc, New Haven, Conn., USA.

A desirable method of modifying DNA encoding the polypeptide of theinvention utilizes the polymerase chain reaction as disclosed by Saikiet al (1988) Science 239, 487-491. This method may be used forintroducing the DNA into a suitable vector, for example by engineeringin suitable restriction sites, or it may be used to modify the DNA inother useful ways as is known in the art.

If viral vectors are used, pox- or adenovirus vectors are preferred.

The DNA (or in the case of retroviral vectors, RNA) may then beexpressed in a suitable host to produce a polypeptide comprising thepeptide or variant of the invention. Thus, the DNA encoding the peptideor variant of the invention may be used in accordance with knowntechniques, appropriately modified in view of the teachings containedherein, to construct an expression vector, which is then used totransform an appropriate host cell for the expression and production ofthe polypeptide of the invention. Such techniques include thosedisclosed in U.S. Pat. Nos. 4,440,859 issued Apr. 3, 1984 to Rutter etal., 4,530,901 issued Jul. 23, 1985 to Weissman, 4,582,800 issued Apr.15, 1986 to Crowl, 4,677,063 issued Jun. 30, 1987 to Mark et al.,4,678,751 issued Jul. 7, 1987 to Goeddel, 4,704,362 issued Nov. 3, 1987to Itakura et al., 4,710,463 issued Dec. 1, 1987 to Murray, 4,757,006issued Jul. 12, 1988 to Toole, Jr. et al., 4,766,075 issued Aug. 23,1988 to Goeddel et al. and 4,810,648 issued Mar. 7, 1989 to Stalker, allof which are incorporated herein by reference.

The DNA (or in the case of retroviral vectors, RNA) encoding thepolypeptide constituting the compound of the invention may be joined toa wide variety of other DNA sequences for introduction into anappropriate host. The companion DNA will depend upon the nature of thehost, the manner of the introduction of the DNA into the host, andwhether episomal maintenance or integration is desired.

Generally, the DNA is inserted into an expression vector, such as aplasmid, in proper orientation and correct reading frame for expression.If necessary, the DNA may be linked to the appropriate transcriptionaland translational regulatory control nucleotide sequences recognised bythe desired host, although such controls are generally available in theexpression vector. The vector is then introduced into the host throughstandard techniques. Generally, not all of the hosts will be transformedby the vector. Therefore, it will be necessary to select for transformedhost cells. One selection technique involves incorporating into theexpression vector a DNA sequence, with any necessary control elements,that codes for a selectable trait in the transformed cell, such asantibiotic resistance.

Alternatively, the gene for such selectable trait can be on anothervector, which is used to co-transform the desired host cell.

Host cells that have been transformed by the recombinant DNA of theinvention are then cultured for a sufficient time and under appropriateconditions known to those skilled in the art in view of the teachingsdisclosed herein to permit the expression of the polypeptide, which canthen be recovered.

Many expression systems are known, including bacteria (for example E.coli and Bacillus subtilis), yeasts (for example Saccharomycescerevisiae), filamentous fungi (for example Aspergillus), plant cells,animal cells and insect cells. Preferably, the system can be mammaliancells such as colorectal cancer- or glioblastoma cells such as thoseavailable from the ATCC Cell Biology Collection.

A typical mammalian cell vector plasmid is pSVL available fromPharmacia, Piscataway, N.J., USA. An example of an inducible mammalianexpression vector is pMSG, also available from Pharmacia. Useful yeastplasmid vectors are pRS403-406 and pRS413-416 and are generallyavailable from Stratagene Cloning Systems, La Jolla, Calif. 92037, USA.Plasmids pRS403, pRS404, pRS405 and pRS406 are Yeast Integratingplasmids (YIps) and incorporate the yeast selectable markers HIS3, TRP1,LEU2 and URA3. Plasmids pRS413-416 are Yeast Centromere plasmids (Ycps).Other vectors and expression systems are well known in the art for usewith a variety of host cells.

The present invention also relates to a host cell transformed with apolynucleotide vector construct of the present invention. The host cellcan be either prokaryotic or eukaryotic. Bacterial cells may bepreferred prokaryotic host cells in some circumstances and typically area strain of E. coli such as, for example, the E. coli strains DH5available from Bethesda Research Laboratories Inc., Bethesda, Md., USA,and RR1 available from the American Type Culture Collection (ATCC) ofRockville, Md., USA (No ATCC 31343). Preferred eukaryotic host cellsinclude yeast, insect and mammalian cells, preferably vertebrate cellssuch as those from a mouse, rat, monkey or human fibroblastic and coloncell lines. Yeast host cells include YPH499, YPH500 and YPH501, whichare generally available from Stratagene Cloning Systems, La Jolla,Calif. 92037, USA. Preferred mammalian host cells include Chinesehamster ovary (CHO) cells available from the ATCC as CCL61, NIH Swissmouse embryo cells NIH/3T3 available from the ATCC as CRL 1658, monkeykidney-derived COS-1 cells available from the ATCC as CRL 1650 and 293cells, which are human embryonic kidney cells. Preferred insect cellsare Sf9 cells, which can be transfected with baculovirus expressionvectors.

Transformation of appropriate cell hosts with a DNA construct of thepresent invention is accomplished by well known methods that typicallydepend on the type of vector used. With regard to transformation ofprokaryotic host cells, see, for example, Cohen et al. (1972) Proc.Natl. Acad. Sci. USA 69, 2110 and Sambrook et al. (1989) MolecularCloning, A Laboratory Manual, Cold Spring Harbor Laboratory, Cold SpringHarbor, N.Y. Transformation of yeast cells is described in Sherman etal. (1986) Methods In Yeast Genetics, A Laboratory Manual, Cold SpringHarbor, N.Y. The method of Beggs (1978) Nature 275, 104-109 is alsouseful. With regard to vertebrate cells, reagents useful in transfectingsuch cells, for example calcium phosphate and DEAE-dextran or liposomeformulations, are available from Stratagene Cloning Systems, or LifeTechnologies Inc., Gaithersburg, Md. 20877, USA. Electroporation is alsouseful for transforming and/or transfecting cells and is well known inthe art for transforming yeast cell, bacterial cells, insect cells andvertebrate cells.

Successfully transformed cells, i.e. cells that contain a DNA constructof the present invention, can be identified by well known techniquessuch as PCR. Alternatively, the presence of the protein in thesupernatant can be detected using antibodies.

It will be appreciated that certain host cells of the invention areuseful in the preparation of the peptides of the invention, for examplebacterial, yeast and insect cells. However, other host cells may beuseful in certain therapeutic methods. For example, antigen-presentingcells, such as dendritic cells, may be used to express the peptides ofthe invention so that they may be loaded into appropriate MHC molecules.Thus, the present invention provides a host cell comprising a nucleicacid or an expression vector according to the invention.

In a preferred embodiment the host cell is an antigen presenting cell(APC), in particular a dendritic cell or antigen presenting cell. APCsloaded with a recombinant fusion protein containing prostatic acidphosphatase (PAP) are currently under investigation for the treatment ofprostate cancer (Sipuleucel-T) (Small E J, et al., J Clin Oncol. 2006;24(19):3089-3094; Rini B I, et al., Cancer. 2006; 107(1):67-74).

A further aspect of the invention provides a method of producing apeptide or its variant. The method comprises culturing the host cell andisolating the peptide from the host cell or its culture medium.

In another embodiment the peptide, the nucleic acid or the expressionvector of the invention are used in medicine. For example, the peptideor its variant may be prepared for intravenous (i.v.) injection,sub-cutaneous (s.c.) injection, intradermal (i.d.) injection,intraperitoneal (i.p.) injection, intramuscular (i.m.) injection.Preferred routes of peptide injection are s.c., i.d., i.p., i.m., andi.v. Preferred routes of DNA injection are i.d., i.m., s.c., i.p. andi.v. Doses of between 50 μg and 1.5 mg, preferably 125 μg to 500 μg, ofpeptide or DNA may be given and will depend from the respective peptideor DNA. Doses of this range were successfully used in previous trials(Brunsvig P F, et al., 2006; 55(12):1553-1564; M. Staehler, et al., ASCOmeeting 2007; Abstract No 3017).

An important aspect of the present invention is an in vitro method forproducing activated CTL. The method comprises contacting in vitro CTLwith antigen loaded human class I or II MHC molecules expressed on thesurface of a suitable antigen-presenting cell for a period of timesufficient to activate the CTL in an antigen specific manner. Theantigen is a peptide according to the invention. Preferably a sufficientamount of the antigen is used with an antigen-presenting cell.

In case of a MHC class II epitope used as an antigen, the CTL areCD4-positive helper cells, preferably of T_(H1)-type. The MHC class IImolecules may be expressed on the surface of any suitable cell andpreferred the cell does not naturally express MHC class II molecules (inwhich case the cell is transfected to express such a molecule).Alternatively, if the cell naturally expresses MHC class II molecules,the cell is defective in the antigen-processing or antigen-presentingpathways. In this way, it is possible for the cell expressing the MHCclass II molecule to be primed substantially completely with a chosenpeptide antigen before activating the CTL.

The antigen-presenting cell (or stimulator cell) typically has MHC classII molecules on its surface and preferably is itself substantiallyincapable of loading said MHC class II molecule with the selectedantigen. The MHC class II molecule may readily be loaded with theselected antigen in vitro.

Preferably the mammalian cell lacks or has a reduced level or hasreduced function of the TAP peptide transporter. Suitable cells whichlack the TAP peptide transporter include T2, RMA-S and Drosophila cells.TAP is the Transporter associated with Antigen Processing.

The human peptide loading deficient cell line T2 is available from theAmerican Type Culture Collection, 12301 Parklawn Drive, Rockville, Md.20852, USA under Catalogue No CRL 1992; the Drosophila cell lineSchneider line 2 is available from the ATCC under Catalogue No CRL19863; the mouse RMA-S cell line is described in Karre and Ljunggren(1985) J. Exp. Med. 162,1745.

It is preferable that the host cell does not express MHC class 1molecules before transfection. Preferably the stimulator cell expressesa molecule important for T-cell costimulation such as any of B7.1, B7.2,ICAM-1 and LFA 3.

The nucleic acid sequences of numerous MHC class II molecules, and ofthe costimulator molecules, are publicly available from the GenBank andEMBL databases.

Similarly, in the case of a MHC class I epitope used as an antigen, theCTL are CD8-positive helper cells. The MHC class 1 molecules may beexpressed on the surface of any suitable cell and it is preferred thatcell does not naturally express MHC class 1 molecules (in which case thecell is transfected to express such a molecule). Alternative, if thecell naturally expresses MHC class 1 molecules, it is defective in theantigen-processing or antigen-presenting pathways. In this way, it ispossible for the cell expressing the MHC class 1 molecule to be primedsubstantially completely with a chosen peptide antigen before activatingthe CTL.

If an antigen-presenting cell is transfected to express such an epitope,preferably the cell comprises an expression vector capable of expressinga peptide containing SEQ ID NO: 1 to SEQ ID NO: 29 or its variant aminoacid sequence.

A number of other methods may be used for generating CTL in vitro. Forexample, the methods described in Peoples et al (1995) Proc. Natl. Acad.Sci. USA 92, 432-436 and Kawakami et al (1992) J. Immunol. 148, 638-643use autologous tumour-infiltrating lymphocytes in the generation of CTL.Plebanski et al (1995) Eur. J. Immunol. 25, 1783-1787 makes use ofautologous peripheral blood lymphocytes (PLBs) in the preparation ofCTL. Jochmus et al (1997) J. Gen. Virol. 78, 1689-1695 describes theproduction of autologous CTL by employing pulsing dendritic cells withpeptide or polypeptide, or via infection with recombinant virus. Hill etal (1995) J. Exp. Med. 181, 2221-2228 and Jerome et al (1993) J.Immunol. 151, 1654-1662 make use of B cells in the production ofautologous CTL. In addition, macrophages pulsed with peptide orpolypeptide, or infected with recombinant virus, may be used in thepreparation of autologous CTL. S. Walter et al., J. Immunol. 2003 Nov.15; 171(10):4974-8 describe the in vitro priming of T-cells by usingartificial antigen presenting cells, which is also a suitable method forgenerating T-cells against the peptide of choice.

Allogeneic cells may also be used in the preparation of CTL and anexemplary method is described in detail in WO 97/26328, incorporatedherein by reference. For example, in addition to Drosophila cells and T2cells, other cells may be used to present antigens such as CHO cells,baculovirus-infected insect cells, bacteria, yeast, vaccinia-infectedtarget cells. In addition, plant viruses may be used (see, for example,Porta et al (1994) Virology 202, 449-955, which describes thedevelopment of cowpea mosaic virus as a high-yielding system for thepresentation of foreign peptides.

The activated CTL that are directed against the peptides of theinvention are useful in therapy. Thus, a further aspect of the inventionprovides activated CTL obtainable by the foregoing methods of theinvention.

Activated CTLs, produced by the above method will selectively recognisea cell that aberrantly expresses a polypeptide comprising an amino acidsequence of SEQ ID NO: 1 to 29.

Preferably, the CTL recognises the cell by interacting through its TCRwith the HLA/peptide-complex (for example, binding). CTLs are useful ina method of killing target cells in a patient wherein the target cellsaberrantly express a polypeptide comprising an amino acid sequence ofthe invention. The patient is administered an effective number of theactivated CTLs. The CTLs administered to the patient may be derived fromthe patient and activated as described above (i.e. they are autologousCTLs). Alternatively, the CTLs are not from the patient but are fromanother individual. Of course, preferably the donor a healthyindividual. By “healthy individual” it is meant that the individual isgenerally in good health, preferably has a competent immune system and,more preferably, is not suffering from any disease that can be readilytested and detected.

The target cells in vivo for the CD4-positive CTL according to thepresent invention can be cells of the tumour (which sometimes expressMHC class II) and/or stromal cells surrounding the tumour (tumour cells)(which sometimes also express MHC class II; (Dengjel, J, et al., 2006,Clin Cancer Res., 12, 4163-4170)).

The CTLs of the invention may be used as active ingredients in atherapeutic composition. Thus, the invention also provides a method ofkilling target cells in a patient where the target cells aberrantlyexpress a polypeptide comprising an amino acid sequence of theinvention. The method comprises administering to the patient aneffective number of CTLs as defined above.

By “aberrantly expressed” we include the meaning that the polypeptide isover-expressed compared to normal levels of expression or that the geneis silent in the tissue from which the tumour is derived but in thetumour it is expressed. By “over-expressed” we mean that the polypeptideis present at a level at least 1.2× that present in normal tissue;preferably at least 2× and more preferably at least 5× or 10× the levelpresent in normal tissue.

CTLs may be obtained by methods known in the art, e.g. those describedabove.

Protocols for this so-called adoptive transfer of CTL are well known inthe art and can be found, e.g. in (Rosenberg, S A, et al., 1987, N.Engl. J. Med., 316, 889-897; Rosenberg, S A, et al., 1988, N. Engl. J.Med, 319, 1676-1680; Dudley, M E, et al., 2002, Science, 298, 850-854;Yee, C, et al., 2002, Proc. Natl. Acad. Sci. U.S.A, 99, 16168-16173;Dudley, M E, et al., M M, 2005, J. Clin. Oncol., 23, 2346-2357);reviewed in Nat. Rev. Immunol., 6, 383-393) and (Morgan, R A, et al.,2006, Science) 314 (5796):126-129).

Any molecule of the invention, i.e. the peptide, nucleic acid,expression vector, cell, activated CTL, T-cell receptor or the nucleicacid encoding it is useful for the treatment of disorders, characterisedby cells escaping an immune response. Therefore, any molecule of thepresent invention may be used as medicament or in the manufacture of amedicament. The molecule may be used by itself or combined with othermolecule(s) of the invention or (a) known molecule(s).

Preferably the medicament is a vaccine. It may be administered directlyinto the patient, into the affected organ or systemically, or applied exvivo to cells derived from the patient or a human cell line which aresubsequently administered to the patient, or used in vitro to select asubpopulation from immune cells derived from the patient, which are thenre-administered to the patient. If the nucleic acid is administered tocells in vitro, it may be useful for the cells to be transfected so asto co-express immune-stimulating cytokines, such as interleukin-2 Thepeptide may be substantially pure, or combined with animmune-stimulating adjuvant (see below) or used in combination withimmune-stimulatory cytokines, or be administered with a suitabledelivery system, for example liposomes. The peptide may also beconjugated to a suitable carrier such as keyhole limpet haemocyanin(KLH) or mannan (see WO 95/18145 and Longenecker et al (1993) Ann. NYAcad. Sci. 690, 276-291). The peptide may also be tagged, or be a fusionprotein, or be a hybrid molecule. The peptides of the present inventionare expected to stimulate CD4 or CD8 CTL. However, stimulation is moreefficient in the presence of help provided by T-cells positive for theopposite CD. Thus, for MHC Class II epitopes that stimulate CD4 CTLm thefusion partner or sections of a hybrid molecule suitably provideepitopes that stimulate CD8-positive T-cells. On the other hand, for MHCClass I epitopes that stimulate CD8 CTLm the fusion partner or sectionsof a hybrid molecule suitably provide epitopes that stimulateCD4-positive T-cells. CD4- and CD8-stimulating epitopes are well knownin the art and include those identified in the present invention.

In one aspect of the invention, the vaccine comprises at least onepeptide, preferably two to 50, more preferably two to 25, even morepreferably two to 15 and most preferably two, three, four, five, six,seven, eight, nine, ten, eleven, twelve or thirteen peptides of theinvention or additional peptides. The peptide(s) may be derived from oneor more specific TAAs and may bind to MHC class I and/or class IImolecules.

Preferably when the peptides of the invention are used in a vaccine ormedicament of the invention, they are present as a salt, such as forexample, but not limited to an acetate salt or a chloride salt. Example7 provides studies of a vaccine IMA-910, which contains some of thepeptides of the present invention and describes the preparation of thevaccine using peptides in their salt form and their particle size.

The polynucleotide may be substantially pure, or contained in a suitablevector or delivery system. The nucleic acid may be may be DNA, cDNA,PNA, CNA, RNA or a combination thereof. Methods for designing andintroducing such a nucleic acid are well known in the art. An overviewis provided by e.g. S. Pascolo, Mol Med 2006, 127; 23-40; R. Stan, J Det al., Hematol Oncol Clin North Am 2006, 3; 613-636 or A Mandavi etal., Curr Oncol Rep 2006, 6, 465-472. Polynucleotide vaccines are easyto prepare, but the mode of action of these vectors in inducing animmune response is not fully understood. Suitable vectors and deliverysystems include viral DNA and/or RNA, such as systems based onadenovirus, vaccinia virus, retroviruses, herpes virus, adeno-associatedvirus or hybrids containing elements of more than one virus. Non-viraldelivery systems include cationic lipids and cationic polymers and arewell known in the art of DNA delivery. Physical delivery, such as via a“gene-gun,” may also be used. The peptide or peptide encoded by thenucleic acid may be a fusion protein, for example with an epitope thatstimulates T-cells for the respective opposite CDR, as noted above.

The medicament of the invention may also include one or more adjuvants.Adjuvants are substances that non-specifically enhance or potentiate theimmune response (e.g., immune responses mediated by CTLs and helper-T(T_(H)) cells to an antigen, and would thus be considered useful in themedicament of the present invention. Suitable adjuvants include, but arenot limited to 1018 ISS, aluminium salts, Amplivax, AS15, BCG,CP-870,893, CpG7909, CyaA, dSLIM, flagellin or TLR5 ligands derived fromflagellin, FLT3 ligand, GM-CSF, IC30, IC31, Imiquimod (ALDARA), ImuFactIMP321, Interferon-alpha or -beta, or pegylated derivatives thereof, ISPatch, ISS, ISCOMATRIX, ISCOMs, Juvlmmune, LipoVac, MALP2, MF59,monophosphoryl lipid A, Montanide IMS1312, Montanide ISA 206, MontanideISA 50V, Montanide ISA-51, water-in-oil and oil-in-water emulsions,OK-432, OM-174, OM-197-MP-EC, ONTAK, OspA, PepTel® vector system, PLGmicroparticles, resiquimod, SRL172, Virosomes and other Virus-likeparticles, YF-17D, VEGF trap, R848, beta-glucan, Pam3Cys, Aquila's QS21stimulon, which is derived from saponin, mycobacterial extracts andsynthetic bacterial cell wall mimics, and other proprietary adjuvantssuch as Ribi's Detox, Quil, or Superfos. Adjuvants such as Freund's orGM-CSF are preferred. Several immunological adjuvants (e.g., MF59)specific for dendritic cells and their preparation have been describedpreviously (Dupuis M et al. 1998; Allison 1998). Also cytokines may beused. Several cytokines have been directly linked to influencingdendritic cell migration to lymphoid tissues (e.g., TNF-α), acceleratingthe maturation of dendritic cells into efficient antigen-presentingcells for T-lymphocytes (e.g., GM-CSF, IL-1 and IL-4) (U.S. Pat. No.5,849,589, specifically incorporated herein by reference in itsentirety) and acting as immunoadjuvants (e.g., IL-12, IL-15, IL-23,IL-7, IFN-alpha, IFN-beta) (Gabrilovich et al. 1996).

CpG immunostimulatory oligonucleotides have also been reported toenhance the effects of adjuvants in a vaccine setting. Without beingbound by theory, CpG oligonucleotides act by activating the innate(non-adaptive) immune system via Toll-like receptors (TLR), mainly TLR9.CpG triggered TLR9 activation enhances antigen-specific humoral andcellular responses to a wide variety of antigens, including peptide orprotein antigens, live or killed viruses, dendritic cell vaccines,autologous cellular vaccines and polysaccharide conjugates in bothprophylactic and therapeutic vaccines. More importantly it enhancesdendritic cell maturation and differentiation, resulting in enhancedactivation of T_(H1) cells and strong cytotoxic T-lymphocyte (CTL)generation, even in the absence of CD4 T-cell help. The T_(H1) biasinduced by TLR9 stimulation is maintained even in the presence ofvaccine adjuvants such as alum or incomplete Freund's adjuvant (IFA)that normally promote a T_(H2) bias. CpG oligonucleotides show evengreater adjuvant activity when formulated or co-administered with otheradjuvants or in formulations such as microparticles, nano particles,lipid emulsions or similar formulations, which are especially necessaryfor inducing a strong response when the antigen is relatively weak. Theyalso accelerate the immune response and enabled the antigen doses to bereduced by approximately two orders of magnitude, with comparableantibody responses to the full-dose vaccine without CpG in someexperiments (Arthur M. Krieg, Nature Reviews, Drug Discovery, Jun. 5,2006, 471-484). U.S. Pat. No. 6,406,705 B1 describes the combined use ofCpG oligonucleotides, non-nucleic acid adjuvants and an antigen toinduce an antigen-specific immune response. A commercially available CpGTLR9 antagonist is dSLIM (double Stem Loop Immunomodulator) by Mologen(Berlin, Germany), which is a preferred component of the pharmaceuticalcomposition of the present invention. Other TLR binding molecules suchas RNA binding TLR 7, TLR 8 and/or TLR 9 may also be used.

Other examples for useful adjuvants include, but are not limited tochemically modified CpGs (e.g. CpR, Idera) Poly(I:C), such as AmpliGen,non-CpG bacterial DNA or RNA as well as immunoactive small molecules andantibodies such as cyclophosphamide, sunitinib, Bavacizumab, celebrex,NCX-4016, sildenafil, tadalafil, vardenafil, sorafinib, XL-999,CP-547632, pazopanib, ZD2171, AZD2171, anti-CTLA4 and SC58175, which mayact therapeutically and/or as an adjuvant. The amounts andconcentrations of adjuvants and additives useful in the context of thepresent invention can readily be determined by the skilled artisanwithout undue experimentation.

Preferred adjuvants are dSLIM, BCG, OK432, ALDARA, PeviTer, andJuvlmmune.

Preferably medicaments of the present invention are active againstcancer. The cancer may be non-metastatic or metastatic, in particularcancer of the buccal cavity and pharynx, cancer of the digestive tract,cancer of the colon, rectum, and anus, cancer of the respiratory tract,breast cancer, cancer of the cervix uteri, vagina, and vulva, cancer ofthe uterine corpus and ovary, cancer of the male genital tract, cancerof the urinary tract, cancer of the bone and soft tissue, and kaposisarcoma, melanoma of the skin, eye melanoma, and non-melanoma eyecancer, cancer of the brain and central nervous system, cancer of thethyroid and other endocrine glands, Hodgkin Lymphoma, Non-HodgkinLymphoma, and myeloma. Most preferably the neoplastic disorder treatedby the method of the current invention is colorectal cancer, lungcancer, breast cancer, pancreatic cancer, prostate cancer, gastriccancer, renal cancer, GIST or glioblastoma.

Since the peptides of the invention were isolated from glioblastoma,colorectal, pancreatic, lung, renal or gastric cancer, the medicament ofthe invention will be particularly useful if cancer to be treated isglioblastoma, colorectal, pancreatic, lung, renal or gastric cancer.

In addition to being useful for treating cancer, the peptides of thepresent invention are also useful as diagnostics. Since the peptideswere generated from glioblastoma and since it was determined that thesepeptides are not present in normal tissues, these peptides can be usedto diagnose the presence of a cancer.

The presence of claimed tumor associated peptides (TAA or TUMAPs) ontissue biopsies can assist a pathologist in diagnosis of cancer.Detection of certain TUMAPs by means of antibodies, mass spectrometry orother methods known in the art can tell the pathologist that the tissueis malignant or inflamed or generally diseased. The presence of groupsof TUMAPs can enable classification or subclassification of diseasedtissues.

The detection of TUMAPs on a diseased tissue specimen can enable thedecision about the benefit of therapies involving the immune system,especially if T lymphocytes are known or expected to be involved in themechanism of action. Loss of MHC expression is a well describedmechanism by which infected of malignant cells escapeimmunosurveillance. Thus, presence of TUMAPs shows that this mechanismis not exploited by the analyzed cells.

TUMAPs might be used to analyze lymphocyte responses against thoseTUMAPs such as T cell responses or antibody responses against the TUMAPor the TUMAP complexed to MHC molecules. These lymphocyte responses canbe used as prognostic markers for decision on further therapy steps.These responses can also be used as surrogate markers in immunotherapyapproaches aiming to induce lymphocyte responses by different means,e.g. vaccination of protein, nucleic acids, autologous materials,adoptive transfer of lymphocytes. In gene therapy settings, lymphocyteresponses against TUMAPs can be considered in the assessment of sideeffects. Monitoring of lymphocyte responses might also be a valuabletool for follow-up examinations of transplantation therapies, e.g. forthe detection of graft versus host and host versus graft diseases.

TUMAPs can be used to generate and develop specific antibodies againstMHC/TUMAP complexes. These can be used for therapy, targeting toxins orradioactive substances to the diseased tissue. Another use of theseantibodies can be targeting radionuclides to the diseased tissue forimaging purposes such as PET. This use can help to detect smallmetastases or to determine the size and precise localization of diseasedtissues.

In addition, the peptides of the invention can be used to verify apathologist's diagnosis of a cancer based on a biopsied sample.

The present invention includes a kit comprising: (a) a container thatcontains a pharmaceutical composition as described above, in solution orin lyophilized form; (b) optionally a second container containing adiluent or reconstituting solution for the lyophilized formulation; and(c) optionally, instructions for (i) use of the solution or (ii)reconstitution and/or use of the lyophilized formulation. The kit mayfurther comprise one or more of (iii) a buffer, (iv) a diluent, (v) afilter, (vi) a needle, or (v) a syringe. The container is preferably abottle, a vial, a syringe or test tube; and it may be a multi-usecontainer. The pharmaceutical composition is preferably lyophilized.

Kits of the present invention preferably comprise a lyophilizedformulation of the present invention in a suitable container andinstructions for its reconstitution and/or use. Suitable containersinclude, for example, bottles, vials (e.g. dual chamber vials), syringes(such as dual chamber syringes) and test tubes. The container may beformed from a variety of materials such as glass or plastic. Preferablythe kit and/or container contains instructions on or associated with thecontainer that indicates directions for reconstitution and/or use. Forexample, the label may indicate that the lyophilized formulation is toreconstituted to peptide concentrations as described above. The labelmay further indicate that the formulation is useful or intended forsubcutaneous administration.

The container holding the formulation may be a multi-use vial, whichallows for repeat administrations (e.g., from 2-6 administrations) ofthe reconstituted formulation. The kit may further comprise a secondcontainer comprising a suitable diluent (e.g., sodium bicarbonatesolution).

Upon mixing of the diluent and the lyophilized formulation, the finalpeptide concentration in the reconstituted formulation is preferably atleast 0.15 mg/mL/peptide (=75 μg) and preferably not more than 3mg/mL/peptide (=1500 μg). The kit may further include other materialsdesirable from a commercial and user standpoint, including otherbuffers, diluents, filters, needles, syringes, and package inserts withinstructions for use.

Kits of the present invention may have a single container that containsthe formulation of the pharmaceutical compositions according to thepresent invention with or without other components (e.g., othercompounds or pharmaceutical compositions of these other compounds) ormay have distinct container for each component.

Preferably, kits of the invention include a formulation of the inventionpackaged for use in combination with the co-administration of a secondcompound (such as adjuvants (e.g. GM-CSF), a chemotherapeutic agent, anatural product, a hormone or antagonist, a anti-angiogenesis agent orinhibitor, a apoptosis-inducing agent or a chelator) or a pharmaceuticalcomposition thereof. The components of the kit may be pre-complexed oreach component may be in a separate distinct container prior toadministration to a patient. The components of the kit may be providedin one or more liquid solutions, preferably, an aqueous solution, morepreferably, a sterile aqueous solution. The components of the kit mayalso be provided as solids, which may be converted into liquids byaddition of suitable solvents, which are preferably provided in anotherdistinct container.

The container of a therapeutic kit may be a vial, test tube, flask,bottle, syringe, or any other means of enclosing a solid or liquid.Usually, when there is more than one component, the kit will contain asecond vial or other container, which allows for separate dosing. Thekit may also contain another container for a pharmaceutically acceptableliquid. Preferably, a therapeutic kit will contain an apparatus (e.g.,one or more needles, syringes, eye droppers, pipette, etc.), whichenables administration of the agents of the invention that arecomponents of the present kit.

The present formulation is one that is suitable for administration ofthe peptides by any acceptable route such as oral (enteral), nasal,ophthal, subcutaneous, intradermal, intramuscular, intravenous ortransdermal. Preferably the administration is s.c., and most preferably,i.d. Administration may be by infusion pump.

For the purposes of the present invention, all references cited hereinare incorporated by reference in their entireties.

EXAMPLES Example 1 Identification of Tumour Associated Peptides (TUMAPS)Presented on Cell Surface

Tissue Samples

Patients' tumour and healthy tissues were provided by several differentclinical sites (see Table below). Written informed consents of allpatients had been given before surgery. Tissues were shock-frozen inliquid nitrogen immediately after surgery and stored until isolation ofTUMAPs at −80° C.

Isolation of HLA Peptides from Tissue Samples

HLA peptide pools from shock-frozen tissue samples were obtained byimmune precipitation from solid tissues according to a slightly modifiedprotocol (Falk, K., et al. Nature 351, 290-296 (1991); Seeger, F. H. etal., Immunogenetics 49, 571-576 (1999)) using the HLA-A*02-specificantibody BB7.2 or the HLA-A, -B, —C-specific antibody W6/32,CNBr-activated sepharose, acid treatment and ultrafiltration.

Detection of TUMAPs by ESI-liquid Chromatography Mass Spectrometry(ESI-LCMS)

The obtained HLA peptide pools were separated according to theirhydrophobicity by reversed-phase chromatography (CapLC, Waters) and theeluting peptides were analyzed in a hybrid quadrupole orthogonalacceleration time of flight tandem mass spectrometer (Q-TOF Ultima,Waters) equipped with an ESI source. Peptide pools were loaded onto aC18 pre-column for concentration and desalting. After loading, thepre-column was placed in line for separation by a fused-silicamicro-capillary column (75 μm i.d.×250 mm) packed with 5 μm C18reversed-phase material (Dionex). Solvent A was 4 mM ammoniumacetate/water. Solvent B was 2 mM ammonium acetate in 80%acetonitrile/water. Both solvents were adjusted to pH 3.0 with formicacid. A binary gradient of 15% to 60% B within 90 minutes was performed,applying a flow rate of 5 μl/min reduced to approximately 200 nl/min bya split-system. A gold coated glass capillary (PicoTip, New Objective)was used for introduction into the micro-ESI source. The integrationtime for the TOF analyzer was 1.9 s with an interscan delay of 0.1 s.Subsequently, the peptide sequences were revealed by collisionallyinduced decay (CID) mass spectrometry (ESI-LCMS/MS). The identifiedTUMAP sequence was assured by comparison of the generated natural TUMAPfragmentation pattern with the fragmentation pattern of a syntheticsequence-identical reference peptide.

FIG. 1 and FIG. 2 show exemplary spectra obtained from tumour tissue forMHC class I associated TUMAPs (FIG. 1 a-1 h) and MHC class II associatedTUMAPs (FIG. 2 a-2 f).

Example 2 Expression Profiling of Genes Encoding the Peptides of theInvention

The peptides identified as being presented on the surface of tumourcells by MHC molecules are likely able to induce T-cells with a highspecificity of recognition for the tissue from which they were derived.In order to minimise the risk for autoimmunity induced by vaccinationwith such peptides the inventors focused on those peptides that arederived from proteins that are overexpressed on tumour cells compared tothe majority of normal tissues.

The ideal peptide will be derived from a protein that is unique to thetumour and not present in any other tissue. To identify peptides thatare derived from genes with an ideal expression profile, the identifiedpeptides were assigned to the proteins and genes, respectively, fromwhich they were derived and expression profiles of the genes weregenerated.

RNA Sources and Preparation

Surgically removed tissue specimens were provided by several differentclinical sites (see Table 2) after written informed consent had beenobtained from each patient.

Tumour tissue specimens were snap-frozen in liquid nitrogen immediatelyafter surgery and later homogenized with mortar and pestle under liquidnitrogen. Total RNA was prepared from these samples using TRIzol(Invitrogen, Karlsruhe, Germany) followed by a cleanup with RNeasy(QIAGEN, Hilden, Germany); both methods were performed according to themanufacturer's protocol.

Total RNA from healthy human tissues was obtained commercially (Ambion,Huntingdon, UK; Clontech, Heidelberg, Germany; Stratagene, Amsterdam,Netherlands; BioChain, Hayward, Calif., USA). The RNA from severalindividuals (between 2 and 123 individuals) was mixed such that RNA fromeach individual was equally weighted. Leukocytes were isolated fromblood samples of 4 healthy volunteers.

Quality and quantity of all RNA samples were assessed on an Agilent 2100Bioanalyzer (Agilent, Waldbronn, Germany) using the RNA 6000 PicoLabChip Kit (Agilent).

Microarray Experiments

Gene expression analysis of all tumour and normal tissue RNA samples wasperformed by Affymetrix Human Genome (HG) U133A or HG-U133 Plus 2.0oligonucleotide microarrays (Affymetrix, Santa Clara, Calif., USA). Allsteps were carried out according to the Affymetrix manual). Briefly,double-stranded cDNA was synthesized from 5-8 μg of total RNA, usingSuperScript RTII (Invitrogen) and the oligo-dT-T7 primer (MWG Biotech,Ebersberg, Germany) as described in the manual. In vitro transcriptionwas performed with the BioArray High Yield RNA Transcript Labelling Kit(ENZO Diagnostics, Inc., Farmingdale, N.Y., USA) for the U133A arrays orwith the GeneChip IVT Labelling Kit (Affymetrix) for the U133 Plus 2.0arrays, followed by cRNA fragmentation, hybridization, and staining withstreptavidin-phycoerythrin and biotinylated anti-streptavidin antibody(Molecular Probes, Leiden, Netherlands). Images were scanned with theAgilent 2500A GeneArray Scanner (U133A) or the Affymetrix Gene-ChipScanner 3000 (U133 Plus 2.0), and data were analysed with the GCOSsoftware (Affymetrix), using default settings for all parameters. Fornormalization, 100 housekeeping genes provided by Affymetrix were used.Relative expression values were calculated from the signal log ratiosgiven by the software and the normal sample was arbitrarily set to 1.0.

Expression profiles of all peptides of the present invention show a highexpression of the respective gene in tumour tissue while being not or toa very low extend expressed in normal tissues.

FIG. 3 shows such profiles for the genes of glioblastoma specificpeptides PTP-001 (gene: PTPRZ1, FIG. 3 a), and CHI-001 (gene: CH3L2,FIG. 3 b).

Example 3 Re-Detection of Identified TUMAPs by ESI-Liquid ChromatographyMass Spectrometry (ESI-LCMS) in Additional Tumour Samples

TUMAPs identified by the method of EXAMPLE 1 were systematicallysearched for on colorectal tumour samples by mass spectrometry.

The obtained HLA peptide pools were separated according to theirhydrophobicity by reversed-phase chromatography (CapLC, Waters) and theeluting peptides were analyzed in a hybrid quadrupole orthogonalacceleration time of flight tandem mass spectrometer (Q-TOF Ultima,Waters) equipped with an ESI source. Peptide pools were loaded onto aC18 pre-column for concentration and desalting. After loading, thepre-column was placed in line for separation by a fused-silicamicro-capillary column (75 μm i.d.×250 mm) packed with 5 μm C18reversed-phase material (Dionex). Solvent A was 4 mM ammoniumacetate/water. Solvent B was 2 mM ammonium acetate in 80%acetonitrile/water. Both solvents were adjusted to pH 3.0 with formicacid. A binary gradient of 15% to 60% B within 90 minutes was performed,applying a flow rate of 5 μl/min reduced to approximately 200 nl/min bya split-system. A gold coated glass capillary (PicoTip, New Objective)was used for introduction into the micro-ESI source. The integrationtime for the TOF analyzer was 1.9 s with an interscan delay of 0.1 s.For detection of defined peptides high sensitive screening was performedin this type of ESI-LCMS experiments on the basis of known molecularweights and retention times of the peptides in the chromatographicsystem. Therefore, a list containing the m/z values of the previouslyidentified peptides (singly and/or doubly charged) was applied forprecursor selection. Subsequently, the sequence was revealed bycollisionally induced decay (CID) mass spectrometry (ESI-LCMS/MS). TheTUMAP sequence was confirmed by comparison of the generated naturalTUMAP fragmentation pattern with the fragmentation pattern of asynthetic sequence-identical reference peptide. Evaluation of the HLApeptide purification yield and reproducibility of the analytical system,including retention time stability was carried out using the intensityand retention time of an abundant endogenous HLA-A*02 peptide (YLLPAIVHIderived from DDX5) as internal standard. Therefore, the CRC sampleinclusion criterion for detection of previously identified TUMAP inthese experiments was set to a minimal intensity of 650 counts per scanof the internal doubly charged standard signal (YLLPAIVHI) in theLCMS/MS experiment to assure a successful HLA peptide isolation and thecorrect performance of the analytical system.

Table 2 shows the results of an analysis of colon and rectum cancersamples of different stages as well as metastases originating fromeither primary tumour site. All HLA-A*02 TUMAPs were found on themajority of samples. Re-detection frequencies of HLA-DR TUMAPs aregenerally lower. This can be expected because for HLA class II peptides,several length variants for each core sequence may exist. ODC-001, aTUMAP identified previously (M Diehl, PhD thesis 1998, University ofTuebingen) and known to be presented on a large number of colon tumoursserved as positive control.

TABLE 2 Re-detection of TUMAPS in CRC samples TUMAP re-detected (+) ornot detected (−) class I class II CRC Tumor Tumor C20- TGFBI- TOP- NOX-PCN- ODC- TGFBI- No sample location stage 001 001 001 001 001 001 004 1CCA062 colon I n.a. n.a. n.a. n.a. n.a. n.a. − 2 CCA740 colonII + + + + + + n.a. 3 CCA165 colon II + + + + + + − 4 CCA712 colonIII + + + − − + n.a. 5 CCA707 colon III + + + + + + n.a. 6 CCA718 colonIII + + + + + + n.a. 7 CCA739 colon III + + + + + + n.a. 8 CCA166 colonIII + + + + + + − 9 CCA734 colon III + + + + + + n.a. 10 CCA719 colonIV + + + + − + n.a. 11 CCA725 colon IV + + + − + + n.a. 12 CCA164 colonIV + + − − + + − 13 CCA167 colon IV n.a. n.a. n.a. n.a. n.a. n.a. − 14CCA056 colon ? n.a. n.a. n.a. n.a. n.a. n.a. − 15 CCA305 colon ? n.a.n.a. n.a. n.a. n.a. n.a. − 20 CCA708 colon IV + + + + + + + metastasis16 CCA160 rectum II + + + + + + + 17 CCA754 rectum II + + + − + + n.a.18 CCA170 rectum III n.a. n.a. n.a. n.a. n.a. n.a. + 19 CCA171 rectum IVn.a. n.a. n.a. n.a. n.a. n.a. − 21 CCA724 rectum IV + + − − − + +metastasis Detected in % of 100% 100% 87% 67% 80% 100% 33% analyzedsamples n.a.: not analysed

Example 4 Binding of HLA Class I-Restricted Peptides to HLA-A*0201

The HLA binding assay was performed using the ELISA EpI Kit (obtainedfrom Sceren Buus, Institute of Medical Microbiology and Immunology atthe University of Copenhagen, Denmark) according to Sylvester-Hvid(Sylvester-Hvid, at al., 2002, Tissue Antigens, 59, 251-258) and theELISA EpI Kit manual by the manufacturer.

Preparation of Peptide Solutions

Peptides were dissolved in DMSO+0.5% TFA (Merck, Darmstadt, Germany) ata concentration of 10 mg/ml. The highest peptide working solution usedin this assay was 200 μM, therefore the stock solution was diluted 1:50in a peptide-dilution buffer (PBS with 0.1% Lutrol-F68 and 10 mg/lPhenol red) to a final volume of 100 μl. A serial five-fold dilution wasperformed with peptide-dilution buffer.

Refolding of HLA-A*0201/Peptide Complexes

According to the manual, a 2-fold concentrated HLA-A*0201 solution wasprepared by mixing 3× pH buffer (pH 6.6), Lutrol-F68, human β2m,recombinant HLA-A*0201 (all included in the ELISA EpI Kit) with PBS.

For the refolding process, 15 μl of peptide serial dilutions and 15 μlof the 2-fold concentrated MHC mix were mixed in 96-well plates (Nunc,Rochester, N.Y., USA) and incubated at 18° C. for 48 hours.

Quantification of the Complexes by an ELISA

Maxisorp plates (Nunc, Rochester, N.Y.) were coated with 5 μg/ml w6/32antibody in coating buffer (pH 9.6), incubated for 24 h at 4° C. andblocked with 5% skim milk powder (Merck, Darmstadt, Germany) in PBS overnight at 4° C.

MHC complex standard (ELISA EpI Kit) was diluted with 2% skim milkpowder in PBS (SMP/PBS) to a concentration of 10 nM. A serial 3.16-folddilution was prepared and transferred to the coated and blocked Maxisorpplate. The peptide-MHC complexes were diluted 10-fold with 2% SMP/PBS,transferred to the same Maxisorp plate and incubated for 2 hours at 4°C. Rabbit anti-hβ2m antibody (ELISA EpI Kit) was added in a 1:2500dilution in 2% SMP/PBS and incubated for 1 hour at 4° C. Amplificationbuffer (HRP-conjugated goat anti-rabbit polymer) and mouse serum (bothsupplied with the ELISA EpI Kit) was diluted in 2% SMP/PBS, added to theplates and incubated 30 minutes at room temperature. Development buffer(Tetramethylbenzidine, TMB; ELISA EpI Kit) was added, plates wereincubated under light protection for 30 minutes at room temperature. Thereaction was stopped by adding 0.2 M sulfuric acid (VWR, Darmstadt,Germany). Plates were read at OD450 nm using the VERSAmax ELISA-Reader(Molecular Devices, Sunnyvale, Calif., USA). Data were interpreted withExcel and Prism®, Graphpad 3.0. Results are shown in FIG. 4. A lower KDvalue reflects higher affinity to HLA-A*0201. Binding affinities stretchover a range of approximately four decades but most peptides havesimilar binding affinities within one decade (C20-001, ODC-001, PCN-001,TOP-001). The affinity of MUC-001 is about one decade lower compared tothe majority of the included ligands but MUC-001 was nevertheless ableto induce a T-cell response when used in a vaccine for renal carcinoma(Wierecky, J, et al., 2006, Cancer Res., 66, 5910-5918). On the otherhand, NOX-001 has a slightly higher binding affinity and TGFBI-001 isthe strongest binder with a 100-fold lower KD value compared with themajority of peptides.

In absolute terms, KD values between 0.01 and 0.1 nM as observed for themajority of peptides represent already a strong binding. Similaraffinities had been also observed for peptides contained in the renalcell carcinoma vaccine IMA901 that was successfully tested (H.Singh-Jasuja, et al., ASCO Meeting 2007 Poster #3017; M. Staehler, etal., ASCO meeting 2007; Poster #3017). Therefore, binding properties ofpeptides of the present invention are quite similar to those of peptidesthat have been shown in vivo to induce a T-cell response.

Example 5 In Vitro Immunogenicity of MHC Class I Presented Peptides

In Vitro Priming of CD8+ T Cells

To perform in vitro stimulations by artificial antigen presenting cells(aAPC) loaded with peptide-MHC complex (pMHC) and anti-CD28 antibody,first PBMCs (peripheral blood mononuclear cells) were isolated fromfresh HLA-A*02+ buffy coats by using standard density gradientseparation medium (PAA, Cölbe, Germany). Buffy coats were eitherobtained from the Blood Bank Tubingen or from the KatharinenhospitalStuttgart. Isolated PBMCs were incubated overnight in T-cell medium(TCM) for human in vitro priming consisting of RPMI-Glutamax(Invitrogen, Karlsruhe, Germany) supplemented with 10% heat inactivatedhuman AB serum (PAA, Cölbe, Germany), 100 U/ml Penicillin/100 μg/mlStreptomycin (Cambrex, Verviers, Belgium), 1 mM sodium pyruvate (CC Pro,Neustadt, Germany) and 20 μg/ml Gentamycin (Cambrex). CD8+ lymphocyteswere isolated using the CD8+ MACS positive selection kit (Miltenyi,Bergisch Gladbach, Germany) according to the manufacturer'sinstructions. Obtained CD8+ T-cells were incubated until use in TCMsupplemented with 2.5 ng/ml IL-7 (PromoCell, Heidelberg, Germany) and 10U/ml IL-2 (Chiron, Munich, Gemany). Generation of pMHC/anti-CD28 coatedbeads, T-cell stimulations and readout was performed as described before(Walter, S, et al., 2003, J. Immunol., 171, 4974-4978) with minormodifications. Briefly, biotinylated recombinant HLA-A*0201 moleculeslacking the transmembrane domain and being biotinylated at the carboxyterminus of the heavy chain were produced following a method describedby (Altman, J D, et al., 1996, Science, 274, 94-96). The purifiedcostimulatory mouse IgG2a anti human CD28 Ab 9.3 (Jung, G, Ledbetter, JA, and Muller-Eberhard, H J; 1987, Induction of cytotoxicity in restinghuman T lymphocytes bound to tumor cells by antibody heteroconjugates,Proc Natl Acad Sci USA, 84, 4611-4615) was chemically biotinylated usingSulfo-N-hydroxysuccinimidobiotin as recommended by the manufacturer(Perbio, Bonn, Germany). Beads used were 5.60 μm large streptavidincoated polystyrene particles (Bangs Labooratories, Illinois/USA). pMHCwas used as positive control and negative controls were A*0201/MLA-001(peptide ELAGIGILTV from modified Melan-A/MART-1) and A*0201/DDX5-001(YLLPAIVHI from DDX5) or A*0201/HBV-001 (FLPSDFFPSV), respectively.

800,000 beads/200 μl were coated in 96-well plates in the presence of600 ng biotin anti-CD28 plus 200 ng relevant biotin-pMHC (high densitybeads) or 2 ng relevant plus 200 ng irrelevant (pMHC library) MHC (lowdensity beads). Stimulations were initiated in 96-well plates byconincubating 1×10⁶ CD8+ T cells with 2×10⁵ washed coated beads in 200μl TCM supplemented with 5 ng/ml IL-12 (PromoCell) for 3-4 days at 37°C. Half of the medium was then exchanged by fresh TCM supplemented with80 U/ml IL-2 and incubation was continued for 3-4 days at 37° C. Thisstimulation cycle was performed for a total of three times. Finally,tetrameric analyses were performed with fluorescent MHC tetramers(produced as described by (Altman, J D, et al., 1996, Science, 274,94-96)) plus antibody CD8-FITC clone SK1 (BD, Heidelberg, Germany) on afour-color FACSCalibur (BD). Peptide specific cells were calculated aspercentage of total CD8+ T cells. Evaluation of tetrameric analysis wasperformed using the software FCS Express (De Novo Software). In vitropriming of specific tetramer+CD8+ lymphocytes was detected byappropriate gating and by comparison to negative control stimulations.Immunogenicity for a given antigen was detected if at least oneevaluable in vitro stimulated well of one healthy donor was found tocontain a specific CD8+ T-cell line after in vitro stimulation (i.e.this well contained at least 1% of specific tetramer+ among CD8+ T-cellsand the percentage of specific tetramer+ cells was at least 10× themedian of the negative control stimulations).

Peptides of the present invention were tested together with peptides ofknown in vivo immunogenicity for comparison. A representative stainingshowing generation of T-cell lines specific for NOX-001 and ODC-001 isshown in FIG. 5. The results are summarized in table 3 below. TheCEA-005 peptide that was optimised for in vitro T-cell induction (Fong,L, et al., 2001, J. Immunol., 166, 4254-4259) served as control. Allpeptides showed in vitro immunogenicity in the PBMCs of healthy donors.

TABLE 3 In vitro immunogenicity of peptides of the invention comparedwith those of vaccine peptides Immunogenicity Antigen detected TGFBI-001yes NOX-001 yes PCN-001 yes TOP-001 yes C20-001 yes ODC-001 yes CCN-001yes PTP-001 yes CHI-001 yes JAK-001 yesTable 4 provides additional in vitro immunogenicity data:

Positive donors/donors Positive wells/wells Antigen tested testedIMA-HBV-001 7/16 (44%)  10/107 (9%) IMA-TGFBI-001  3/4 (75%)  4/22 (18%)IMA-NOX-001  3/5 (60%)  9/60 (15%) IMA-PCN-001  3/4 (75%)  4/42 (10%)IMA-TOP-001  2/5 (40%)  7/72 (10%) IMA-C20-001  1/5 (20%)  1/60 (2%)IMA-ODC-001  1/5 (20%)  1/60 (2%) IMA-HBV-001  2/5 (40%)  10/54 (19%)IMA-CEA-004  4/4 (100%)  50/60 (83%) IMA-CCN-001  5/5 (100%)  42/54(78%) IMA-MET-001  4/6 (67%)  30/72 (42%)

Results of in vitro immunogenicity experiments conducted by immatics aresummarised here. Results shown have been obtained by stimulation of CD8+cells with high density beads. As different human serum lots may highlyaffect the immunogenicity results, only assays in which one and the sameserum lot was used, were evaluated together.

Example 6 In Vitro Immunogenicity for MHC Class II Presented Peptides

T helper cells play an important role in supporting CTLs to activate andsustain immune responses against tumor cells. Therefore, MHC class IIpeptides were included in IMA910. TGFBI-004, one of the three class IIpeptides contained in IMA910, was tested for its immunogenic potentialin vitro and proved to be an inducer of both specific CD4+ and CD8+ Tcells. The generation of CD4+ and functional CD8+ T lymphocytes wasshown in experiments using stimulations performed in an autologoussystem.

Principle of Test

Priming and expansion of specific human CD4+ and CD8+ cells were assayedin vitro by priming of monocyte-depleted PBMCs with autologous DCs andrestimulation with autologous PBMCs. Briefly, to generateantigen-specific CD4+ T cells, monocyte-depleted PBMCs of one healthydonor (HLA genotype class I: A1/A25/B8/B18 and class II:DQB1*02/DQB1*06/DRB1*03/DRB1*15/DRB3/DRB5) were stimulated usingpeptide-pulsed autologous DCs and restimulated with autologous PBMCsplus peptide. As a read-out system, IFNγ production upon short termrestimulation was assessed by ELISPOT and flow cytometry. T cells wereanalysed after eight stimulations by ELISPOT and intracellular IFNγstaining plus CD4-FITC and CD8-PerCP to determine the percentage ofIFNγ-producing cells in specific T-cell subpopulations. In thisexperiment, cells stimulated with TGFBI-004 peptide from different wellswere pooled, incubated with irrelevant peptide for the read-out andperformed as negative controls.

Generation of Dendritic Cells (DCs)

Human DCs were obtained from monocytes cultured in DC medium consistingof RPMI 1640-Glutamax/25 mM Hepes (Invitrogen, Germany) supplementedwith 10% autologous plasma//100 U/ml penicillin and 100 μg/mlstreptomycin. First, buffy coat and plasma was obtained bycentrifugation of the blood from a healthy donor (Bloodbank Tubingen).PBMCs were then isolated from the buffy coat by standard densitygradient separation (Lymphocyte Separation Medium, PAA, Austria) andresuspended in DC medium to determine total cell number. 100-120 Millionof PBMCs were washed, resuspended in 15 ml X-Vivo 20 medium(BioWhittaker, Belgium) and transferred to a cell culture flask. After 2hours at 37° C., media containing peripheral blood leukocytes (PBL) wasremoved, adherent monocytes were washed twice with 10 ml PBS andcultured for 6 days in 10 ml DC medium with 100 ng/ml GM-CSF and 30ng/ml IL-4 (ImmunoTools, Germany) or 20 ng/ml (R&D systems, Germany). Onday 3 and 5, 100 ng/ml GM-CSF and 30 ng/ml IL-4 (Immunotools) or 20ng/ml IL-4 (R&D Systems, Germany) was added. On day 7 immature DCs wereactivated with 10 ng/ml TNF-α (R&D Systems, Germany) and 20 μg/mlpoly(IC) (Sigma Aldrich, Germany) or 100 ng/ml LPS for 24 hours.Remaining PBMCs and obtained PBLs were aliquoted and frozen.

In Vitro Priming of Specific T Cells

To generate CD4+ T cells, 3 Million PBMCs/PBLs were stimulated with2×10⁵ autologuous DCs. DCs were harvested on day 8 (see chapter 3.1,Generation of DCs). PBS with 5 mM EDTA was used for this purpose inorder to gain as many cells as possible (including adherent cells).After being washed with DC medium, cell number was determined. Forloading with peptide, DCs were resuspended in 1 ml DC medium andincubated with 25 μg/ml peptide for 2 hours at 37° C. Peptides used forpulsing of DCs were TGFBI-004, Posmix (mix of EBV and CMV relatedpeptides), Padre and CMV. Autologous PBMCs/PBLs were thawed, washed withDC medium (at least twice) and plated in a 24 well plate at a density of3 Million cells/ml in 1 ml. DCs loaded with peptide were then added (as1 ml suspension containing the peptide) to the plated PBMCs/PBLs andincubated for 7 days at 37° C. After priming, obtained CTLs were firstrestimulated with cryopreserved autologous peptide-loaded PBMCs whichhave been irradiated (30 Gy; Gammacell 1000 Elite, NordionInternational, Canada). 5×10⁵ CTLs and 2.5×10⁶ PBMCs were added per wellfor this purpose. Pulsing of PBMCs with peptide was performed asaforementioned (for DCs). On day 1 after the first restimulation, IL-2(R&D Systems, Germany) and IL-7 was added to a final concentration of 2ng/ml and 5 ng/ml, respectively. Afterwards, every 2nd day and every 7thday, IL-2 and IL-7 were added to the media. Second restimulation wasperformed 7 days later, but this time peptide was added alone (withoutPBMCs) to the cultured CTLs. Restimulations were performed in a 7 daycycle, with peptide-loaded PBMCs and peptide alone being addedalternatively. Analyses were performed after the eight stimulation byintracellular IFNγ staining and IFNγ ELISPOT.

Results

It was possible to prime CD4+ T cell lines specifically reacting to thepeptide of interest (FIG. 6 and FIG. 3). T-cell responses could bedetected via ELISPOT in 2 out of 4 T-cell lines, whereas in 3 out of 4T-cell lines, TGFBI-004 specific IFNγ producing CD4+ and/or CD8+ cellswere shown via ICS.

Thus, TGFBI-004 was able to elicit CD4+ and CD8+ T cell responses in onedonor tested with the above described experimental system. According tothis promising result, it is likely that this peptide is immunogenic andhas the capacity to induce T-cell responses.

Functional Validation Exemplified by NOX-001 and TGFBI-001

Immunogenicity of peptides included in IMA910 vaccine was demonstratedin vitro by using immatics' TUMAP validation platform. The induction ofspecific T cells is an indication for the ability of peptides tosuccessfully activate the immune system. Since efficient anti-tumorimmune response is only possible when activated T cells are of highavidity and functional, the TUMAPs' ability to prime high avidity,functional T lymphocytes was investigated by testing their ability toproduce IFNγ or to kill tumor cell lines. Two peptides, NOX-001 andTGFBI-001, were chosen for deeper validation due to their capacity toinduce high avidity CTLs in vitro. The results proved that high avidityprecursor T cells exist against both peptides in humans and thatfunctional CD8+ T cell lines could be generated by NOX-001.

Principle of Test

To get additional insight on the immunogenicity of IMA910 peptides andthe properties of specific T cells, two peptides, NOX-001 and TGFBI-001,were selected for further evaluation. The experiments performed for thispurpose were conducted at immatics (cell sorting was performed at theUniversity of Tubingen, lab of Dr. Baring).

Dependent on their ability to be activated by high- or low-densityantigen, T cell lines can be divided into high- or low-avidity. As ithas been shown before (Walter, S, et al., 2003, J. Immunol., 171,4974-4978), human high-avidity CTLs can be raised successfully by usingless peptide for activation compared to low-avidity CD8+ T cells. It hasalso been demonstrated that cells expanded this way are more efficientin recognizing antigen-expressing tumor cell lines, hereby constitutinga possible major tool in the development of therapy strategies.

To determine the ability of peptides to generate high-avidity CTL lines,isolated human CD8+ cells were primed and expanded by repeated in vitrostimulations with beads coated with low-density pMHC(peptide-MHC-complex) and anti-CD28 antibody in the presence of IL-12and IL-2. After three stimulations, a fraction of in vitro primed Tcells were pMHC-tetramer stained and detected by cytometric analysis.Tetramer-positive cells of each donor were pooled afterwards accordingto the antigen specificity, stained with pMHC-tetramer and humananti-CD8-FITC antibody and finally subjected to FACS sorting on aFACSAria. Sorted cells were cultured and expanded in the presence ofirradiated feeder cells, cytokines and mitogen. As a read-out for thegeneration of primed high avidity antigen specific cells, pMHC-tetramerstaining was performed. In order to determine their functionality, IFNγproduction was assayed by ELISPOT and killing of tumor cell lines wasexamined using a cytotoxicity assay based on live/dead staining afterrestimulation of the cells with the corresponding peptide and tumor celllines.

Generation of Specific CD8+ T-cell Lines

In vitro stimulations using artificial antigen presenting cells (aAPC)loaded with peptide-MHC complex (pMHC) and anti-CD28 antibody wereconducted as described above. The only difference to the methoddescribed above was the fact that stimulations were performed with beadsloaded with 2 ng relevant plus 200 ng irrelevant library (pMHC)MHC (lowdensity beads) instead of 200 ng relevant MHC (high density beads).Thus, predominantly high avidity T cells were generated for deepervalidation of peptides. After three stimulations, a fraction of in vitroprimed T cells was pMHC-tetramer stained and detected by cytometricanalysis. Immunogenicity for a given antigen was detected if at leastone evaluable in vitro stimulated well of one healthy donor was found tocontain a specific CD8+ T-cell line after in vitro stimulation (i.e.this well contained at least 1% of specific tetramer+ among CD8+ T-cellsand the percentage of specific tetramer+ cells was at least 10× themedian of the negative control stimulations). Tetramer-positive cells ofeach donor were pooled afterwards according to the antigen specificity,stained with the corresponding pMHC-tetramer and human anti-CD8-FITCantibody clone SK1 and finally subjected to FACS sorting on a FACSAria(BD Biosciences, Germany). Sorted cells were cultured in T cell medium(RPMI-Glutamax supplemented with 10% heat inactivated human AB serum,100 U/ml penicillin, 100 μg/ml streptomycin, 1 mM sodium pyruvate and 20μg/ml Gentamycin) in the presence of 5×10⁵ cells/ml irradiated freshallogeneic PBMCs, 5×10⁴ cells/ml irradiated LG2-EBV cells, 150 U/ml IL-2(Chiron, Munich, Germany) and 0.5 μg/ml PHA-L (Roche Diagnostics,Mannheim, Germany). Expansion of these cells occurred in T cell mediumcontaining 150 U/ml IL-2. As a read-out for the generation of primedhigh avidity antigen specific cells, pMHC-tetramer staining wasperformed as above and analyzed on a four-color FACSCalibur (BDBiosciences, Germany).

Functionality Tests

To determine their functionality, IFNγ production was assessed byELISPOT (IFNγ ELISPOT Set, BD, Germany) after restimulation of the cellswith the corresponding peptide. Additionally, cell-mediated cytotoxicityof specific CTLs was investigated by killing of tumor cell lines usingthe LIVE/DEAD cell-mediated cytotoxicity Kit (L7010, Invitrogen,Germany). Both assays were performed according to manufacturer'sinstructions, except noted otherwise.

Results

Both peptides, NOX-001 and TGFBI-001, were immunogenic in vitro as shownby successful priming with low pMHC density aAPCs. For NOX-001 as wellas for TGFBI-001 specific T-cell lines could be established by FACS,thus demonstrating that high-avidity CD8+ T cell precursors exist inhealthy donors.

Additionally, for NOX-001, one T-cell line could be established thatalso proved to be functional by ELISPOT since it was specificallyexpressing IFNγ after restimulation with this peptide (FIG. 8).

Example 7 Synthesis of a Vaccine Comprising Some of the Peptides of thePresent Invention

Synthesis and Structure

Peptides were synthesized by standard and well-established solid phasesynthesis using Fmoc chemistry. After purification by preparative HPLC,ion-exchange procedure was performed to incorporate physiologicalcompatible counter ions (acetate or chloride). Finally, white to offwhite solids were obtained after lyophilization. All TUMAPs areadministered as acetate salts except IMA-CCN-001 which is supplied aschloride salt for technical reasons during the manufacturing procedure.

Importantly, identity and purity of the peptides can be determinedeasily and with high accuracy using mass spectrometry, amino acidanalysis and analytical HPLC. According to analytical results, allpeptides used for IMA910 vaccine show the correct structure withpurities≧95%.

TABLE 5 Physico-chemical characteristics of peptides in vaccine IMA910Peptide length (no of amino Physical Hygro- No. Peptide ID acids) Saltform form scopicity 1 IMA-C20-001 9 Acetate White Stored as 2IMA-CCN-001 9 Chloride to off- freeze dried 3 IMA-CEA-004 9 Acetatewhite powder. 4 IMA-CEA-006 16 Acetate powder Lyophilized 5 IMA-HBV-00110 Acetate peptides 6 IMA-MET-001 9 Acetate generally 7 IMA-MMP-001 16Acetate have 8 IMA-MUC-001 9 Acetate hygro- 9 IMA-NOX-001 9 Acetatescopic 10 IMA-ODC-001 9 Acetate properties. 11 IMA-PCN-001 10 Acetate 12IMA-TGFBI- 10 Acetate 001 13 IMA-TGFBI- 15 Acetate 004 14 IMA-TOP-001 10Acetate

Particle size distribution and particle shape measurement of theparticles obtained after reconstitution have been performed by capturingdirect images of each individual particle in the range of 0.25 to 100 μmfollowed by image analysis. As a result the majority (>95%) of theparticles have been found in the range of 0.25 to 2.7 μm. So far, nomajor differences in size and shape distribution could be observedwithin 1, 2 or 3 hours after reconstitution.

Furthermore, analytical HPLC was performed for a closer characterizationof the obtained suspension. It could be demonstrated that the particlesconsist mainly of two peptides (IMA-TGFBI-001 and IMA-NOX-001) which arealmost insoluble in the solution used for reconstitution of IMA910.Additionally, low amounts of 4 other peptides (IMA-CEA-006, IMA-TOP-001,IMA-CCN-001 and IMA-HBV-001) were also found in the particles. Thecomposition of the particles (qualitatively and quantitatively) wasfound to be very similar in two independently manufactured batches.

Mannitol and Polysorbate 80 (Tween 80) have been used as excipients andnon-active ingredients to improve solubility characteristics of thepeptide lyophilisate.

IMA910 is dissolved in 700 μL sodium hydrogen carbonate (4.2%).

To reconstitute IMA910, 700 μL of the diluent is injected through thestopper into the vial by a 1 mL syringe equipped with a needle. Todissolve IMA910, the vial and the diluent shall be shaken gently forabout 2 minutes. Shaking should be performed carefully in order to avoidstrong foaming. By this procedure a white to off-white homogeneoussuspension will be obtained. To avoid any sedimentation the vial contentshall be gently shaken before transferring 500 μL of this suspensioninto a new syringe equipped with a needle (size: G20). 10 to 30 minutesafter GM-CSF injection administer 500 μL reconstituted IMA910 i.d. atthe same injection site. Administration has to occur within 1 h afterreconstitution. Dissolved lyophilisate may be stored aseptically at roomtemperature for up to 1 hour following reconstitution.

IMA910 is composed of a cocktail of 13 synthetic tumor-associatedpeptides (TUMAPs) of which the majority has been identified on primarycolorectal carcinoma (CRC) cells. The TUMAPs include 10 HLA classI-binding peptides with the capacity to activate cytotoxic T cells (CD8+T cells) and 3 HLA class II-binding peptides with the capacity toactivate T helper cells (CD4+ T cells). In addition to these 13 TUMAPsIMA910 contains one control peptide of viral origin.

Example 8 Binding of HLA Class I-restricted Peptides of the Invention toHLA-A*0201

The objective of this analysis was to evaluate the affinity of the HLAclass I peptides CHI-001, DCA-001, JAK-001 and PTP-001 to the MHCmolecule coded by the HLA-A*0201 allele. Affinities for all peptides toHLA-A*0201 were comparable to the well-known control peptide HBV-001,dissociations constants (K_(D)) being in the range from 0.05 to 1.6 nM.

Principle of Test

Stable HLA/peptide complexes consist of three molecules: HLA heavychain, beta-2 microglobulin (b2m) and the peptidic ligand. The activityof denatured recombinant HLA-A*0201 heavy chain molecules alone can bepreserved making them functional equivalents of “empty HLA-A*0201molecules.” When diluted into aqueous buffer containing b2m and anappropriate peptide, these molecules fold rapidly and efficiently in anentirely peptide-dependent manner. The availability of these moleculesis used in an ELISA-based assay to measure the affinity of interactionbetween peptide and HLA class I molecule (Sylvester-Hvid C, et al.,(2002) Tissue Antigens 59, 251-258).

Purified recombinant HLA-A*0201 molecules were incubated together withb2m and graded doses of the peptide of interest. The amount of denovo-folded HLA/peptide complexes was determined by a quantitativeELISA. Dissociation constants (K_(D) values) were calculated using astandard curve recorded from dilutions of a calibrant HLA/peptidecomplex.

Results

Results are shown in FIG. 9 A lower K_(D) value reflects higher affinityto HLA-A*0201. Affinities for all peptides to HLA-A*0201 were comparableto the well-known control peptide HBV-001, dissociations constants(K_(D)) being in the range from 0.05 to 1.6 nM.

The invention claimed is:
 1. An isolated peptide comprising SEQ ID No.2, wherein said peptide has an overall length of not more than 100 aminoacids, or a pharmaceutically acceptable salt thereof.
 2. The peptideaccording to claim 1, wherein said peptide has an overall length of from8 to 30 amino acids.
 3. An isolated peptide having an overall length ofnot more than 30 amino acids, wherein said peptide comprises a variantof SEQ ID NO: 2, wherein said variant of SEQ ID No. 2 is modified inthat at least one side chain of one or two amino acid residues arealtered and wherein the peptide and/or variant is able to bind to an HLAmolecule in substantially the same way as a peptide consisting of theamino acid sequence of SEQ ID No. 2, or a pharmaceutically acceptablesalt thereof.
 4. The peptide according to claim 1, wherein the peptideconsists of an amino acid sequence according to SEQ ID No.
 2. 5. Thepeptide according to claim 1, wherein said peptide includes non-peptidebonds.
 6. A fusion protein, comprising SEQ ID NO: 2 and N-terminal aminoacids of the HLA-DR antigen-associated invariant chain (Ii), or apharmaceutically acceptable salt thereof.
 7. A method of producing apeptide and/or variant according to claim 1, the method comprisingculturing a host cell and isolating the peptide from the host celland/or a culture medium thereof.
 8. An in vitro method for producingactivated cytotoxic T lymphocytes (CTL), the method comprisingcontacting in vitro CTL with antigen loaded human class I or II MHCmolecules expressed on a surface of an antigen-presenting cell for aperiod of time sufficient to activate said CTL in an antigen specificmanner, wherein said antigen is a peptide according to claim
 1. 9. Themethod according to claim 8, wherein the antigen is loaded onto a classI or II MHC molecule expressed on a surface of an antigen-presentingcell by contacting said antigen with said antigen-presenting cell. 10.The method according to claim 8, wherein the antigen-presenting cellcomprises an expression vector.
 11. A medicament for inducing an immuneresponse in a subject, said medicament comprising a peptide of claim 1,or a pharmaceutically acceptable salt thereof.
 12. A medicamentaccording to claim 11, wherein the medicament is a vaccine.
 13. Amedicament according to claim 11, wherein the medicament is activeagainst cancer cells.
 14. A medicament according to claim 13, whereinsaid cancer cells comprise glioblastoma cells, colorectal, pancreatic,lung, renal or gastric cancer cells.